CN115927097B - Bacillus bailii FC02, microbial inoculum, preparation method and application thereof - Google Patents

Abstract

The invention relates to the technical field of pesticides and application thereof, in particular to bacillus bailii FC02, a microbial inoculum, a preparation method and application thereof. The invention provides bacillus beleiensis (Bacillus velezensis) FC02, and the preservation number of the bacillus beleiensis FC02 is CGMCC No.22512. The strain can efficiently degrade the paradinitroaniline pesticides, can effectively repair water, soil and crops polluted by the dinitroaniline pesticides, solves the problem of exceeding the standard of dinitroaniline pesticides and the problem of environmental pollution in agricultural production, and protects ecological environment and human health.

Description

Bacillus Velez FC02, bacterial agent, preparation method and application thereof

Technical Field

The invention relates to the field of pesticide technology and application, and in particular to a strain of Bacillus Velezii FC02, a bacterial agent, and a preparation method and application thereof.

Background technique

Dinitroaniline herbicides are selective pre-emergence treatment agents. Butralin, as one of the commonly used dinitroaniline herbicides, is likely to remain in the soil, environment and food after application and cause a certain degree of environmental pollution through migration and penetration, and ultimately pose a potential threat to human health through the food chain. At present, heavy residues of dinitroaniline herbicides are often found in matrices such as fruits and vegetables, meat, water and soil.

Butyl has low solubility in water at room temperature, high octanol-water partition coefficient, low volatility, and long residual period in soil. Under indoor room temperature and aerobic conditions, the time required for the concentration of butyl in the environment to degrade to half of the initial concentration (disappearance time, _DT50 ) is as long as 1949 days, and the _DT50 in the field is 105 days. Butyl is highly toxic to aquatic crustaceans, with an acute toxicity median lethal concentration ( _LC50 ) of 0.061 mg/L in 96 hours; it is moderately toxic to aquatic invertebrates, algae, fish, bees and earthworms, and has a high potential for bioaccumulation. In addition, the storage behavior and residue level of butyl in Solanaceous vegetables in China need to be clarified, which is conducive to the comprehensive evaluation of dietary exposure risks of Chinese residents.

Bioremediation technology based on microorganisms can be used to degrade a variety of organic pollutants. It has the advantages of safety, high efficiency, no secondary pollution, and low cost. It is the forefront and hot spot of international research on residual pesticide treatment technology. The resources of efficient degradation strains for microbial degradation of dinitroaniline herbicides are still scarce. Degradation fungi are mainly concentrated in the genera Penicillium sp., Aspergillus sp. and Fusarium sp., which have good degradation effects on dinitroaniline herbicides. However, reports on the microbial degradation of butralin are very rare, and there are only two cases at present: Ni Haiyan isolated and purified a Bacillus subtilis Y3 that can degrade butralin from activated sludge that has been used to treat pesticide production wastewater for a long time (Ni Haiyan. Isolation and identification of dinitroaniline herbicidebutralin-degrading strains, analysis of degradation pathways and cloning of nitroreductase genes [D]. Nanjing Agricultural University, 2016); Sunil Ghatge isolated and purified a strain of Sphingopyxis sp. HMH from the soil in an agricultural area (Gyeongsan) in South Korea, which can effectively degrade butralin (A, Sunil Ghatge, et al. "A novel pathway for initial biotransformation of dinitroaniline herbicidebutralin from a newly isolated bacterium Sphingopyxis sp. strain HMH-Science Direct." Journal of Hazardous Materials 402(2020).). Therefore, conducting research on microbial remediation technology for butarin and screening for efficient microbial strains that can degrade butarin can provide an effective means to reduce butarin residues in soil, water or crops, and provide technical support for ensuring the safety of agricultural products and farmland environment.

At present, the number of microbial strains with high efficiency in degrading dinitroaniline herbicides, especially butralin, is very limited. Moreover, the mechanism of pesticide degradation using microorganisms is very complex and highly targeted. Therefore, there is an urgent need for a strain with high efficiency in degrading dinitroaniline herbicides, especially butralin.

Summary of the invention

In order to solve the above problems, the present invention provides a strain of Bacillus Velezii FC02, a bacterial agent, and a preparation method and application thereof. The strain of the present invention can efficiently degrade dinitroaniline pesticides, and can also effectively repair water bodies and soils contaminated by dinitroaniline pesticides. Crops can be planted in the repaired soil or water bodies, which also reduces pesticide pollution in crops, solves the problem of excessive dinitroaniline pesticide residues and environmental pollution in agricultural production, and protects the ecological environment and human health.

In order to achieve the above object, the present invention provides the following technical solutions:

The invention provides a strain of Bacillus velezensis FC02, and the deposit number of the Bacillus velezensis FC02 is CGMCC No.22512.

The present invention provides a bacterial agent or disinfectant for degrading dinitroaniline pesticides, wherein the active ingredients of the bacterial agent or disinfectant include the fermentation liquid, seed liquid or bacterial body of Bacillus Velez FC02 described in the above technical scheme.

Preferably, the number of effective live bacteria in the bacterial agent or disinfectant is 18 to 20 billion/g.

Preferably, the bacterial agent or disinfectant further comprises auxiliary materials; the auxiliary materials comprise carriers and auxiliary agents.

Preferably, the volume mass ratio of the active ingredient to the excipient is 200-500 mL:300-600 g.

The present invention provides a method for preparing the bacterial agent or disinfectant described in the above technical solution, comprising the following steps:

After the active ingredients are mixed with auxiliary materials, they are dried to obtain a bacterial agent or disinfectant.

Preferably, the drying is constant temperature drying, the drying temperature is 35-40° C., and the drying time is 8-11 hours.

The present invention provides the use of the Bacillus Velez FC02 described in the above technical solution or the bacterial agent or disinfectant described in the above technical solution or the bacterial agent or disinfectant prepared by the preparation method described in the above technical solution in degrading dinitroaniline pesticides in an environment.

The present invention provides the use of Bacillus Velez FC02 described in the above technical solution, or the bacterial agent or disinfectant described in the above technical solution, or the bacterial agent or disinfectant prepared by the preparation method described in the above technical solution in degrading butralin in an environment.

The present invention provides a method for efficiently degrading dimethylaniline pesticides, comprising the following steps:

The bacterial agent described in the above technical solution or the bacterial agent or disinfectant prepared by the preparation method described in the above technical solution is applied to the area contaminated by dimethylaniline pesticides.

Beneficial effects:

The invention provides a strain of Bacillus velezensis FC02, and the deposit number of the Bacillus velezensis FC02 is CGMCC No. 22512. The strain of the invention can effectively degrade pesticide dinitroaniline pesticide residues, especially butralin residues, in a short time, protect the ecological environment and human health, and is easy to use, low in cost, and has a removal rate of more than 80%. It can be used to repair water bodies, soils, etc. polluted by dinitroaniline pesticides, degrade dinitroaniline pesticide residues on crops, solve the problem of excessive pesticide residues and environmental pollution in agricultural production, and protect the safety of crop production and human health.

The present invention also provides a bacterial agent, which has high active ingredients, excellent product performance indicators, wettability <1min, average suspension rate of 78%, average moisture content of 1.79%, fineness (passing 400 mesh standard sieve) ≥99%, and pH value of 7.8. The bacterial agent has good stability and storage resistance, can be used to degrade butralin residues in water, soil or crops, has the advantages of high efficiency, strong operability, low production cost, and convenient use, has a live bacteria count of 20 billion/g, is suitable for treating pollution caused by butralin in water bodies, soil and other environments, and has very important theoretical and application value.

Biological Deposit Certificate

Bacillus velezensis FC02 was deposited in the General Microbiology Center of China Microorganism Culture Collection (CGMCC) on May 12, 2021. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the deposit number CGMCC No. 22512 and the postal code is 100101.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the degradation of butralin by strains Ⅰ to Ⅳ on a plate;

Figure 2 shows the morphological characteristics of multiple bacterial colonies;

Figure 3 shows the morphological characteristics of a single bacterial colony;

Figure 4 shows the morphological characteristics and length calibration of the strains under a scanning electron microscope;

Figure 5 shows the morphological characteristics of the strain under a scanning electron microscope;

Figure 6 shows the phylogenetic tree of strain II, where FC02 is strain I;

Figure 7 is a color visualization of the degradation of butralin in MSM containing Bacillus Velez FC02 and a color visualization of the degradation of butralin in MSM;

FIG8 is a graph showing the degradation effect of Bacillus Velez FC02 on butralin and the growth of the strain, wherein Residuaiacephate is the residual amount of butralin, time is time, and Cell growth is the cell growth amount;

FIG9 is a graph showing the degradation effect of the bacterial agent on butralin;

FIG10 is a mass spectrum and predicted structure of butralin metabolite A;

FIG11 is a mass spectrum and predicted structure of butralin metabolite B;

FIG12 is a mass spectrum and predicted structure of butralin metabolite C;

FIG13 is a mass spectrum and predicted structure of butralin metabolite D;

FIG14 shows the effect of inoculum size on the degradation of butralin by strain FC02;

FIG15 shows the effect of temperature on the degradation of butralin by strain FC02;

FIG. 16 shows the effect of pH on the degradation of butralin by strain FC02.

Detailed ways

Unless otherwise specified, the components or reagents described in the present invention can be purchased by those skilled in the art.

The present invention provides a strain of Bacillus velezensis FC02, and the deposit number of the Bacillus velezensis FC02 is CGMCC No. 22512. The strain was isolated and preserved from the wooden board of the Southern Song Dynasty shipwreck "Nanhai No. 1", and was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on May 12, 2021. In a specific embodiment of the present invention, the bacterial agent is treated with a high concentration of 200 mg/L of butralin for 6 days, and the degradation rate reaches 89.92%, which can efficiently degrade the butralin residual in the environment, and has the advantages of low production cost, easy use, and good degradation effect.

The single colony morphology of the Velez Bacillus FC02 described in the present invention is nearly round, light yellow and opaque; the colony surface is smooth and has neat edges in the early stage of cultivation, and wrinkles are present on the surface in the later stage, the edges are slightly irregular, there is a bulge in the middle, and the colonies are diffused in a cloud-like manner around; under a transmission electron microscope, the hyphae cell wall and cell membrane of the Velez Bacillus FC02 colony cultured on a NA plate for about 48 hours are closely connected and have a complete structure, the cell matrix is evenly distributed, the cross-section is regular and has a clear structure, and the cell nucleus and other organelles are in a good growth state; under a scanning electron microscope, the strain is rod-shaped, the shape is full, the two ends of the cell are blunt-rounded, there is no capsule, and the size is (1.6-2.2)×(0.68-0.70) microns.

The 16S rDNA nucleotide sequence of the Bacillus Velez FC02 of the present invention is shown in SEQ ID NO: 1. The Bacillus Velez FC02 of the present invention can effectively degrade the pesticide butralin residue in a short time, protect the ecological environment and human health, and is easy to use, low in cost, and has a removal rate of more than 80%. It can be used to repair water bodies and soils contaminated by butralin, degrade butralin residues on crops, solve the problem of excessive pesticide residues and environmental pollution in agricultural production, and protect the safety of crop production and human health.

The present invention provides a bacterial agent or disinfectant for degrading dinitroaniline pesticides, wherein the active ingredient of the bacterial agent or disinfectant includes the fermentation liquid, seed liquid or bacterial body of Bacillus Velez FC02 described in the above technical solution, preferably the fermentation liquid of Bacillus Velez. The dinitroaniline pesticides of the present invention preferably include dinitroaniline pesticide herbicides, more preferably butralin. In the present invention, the effective viable count of the bacterial agent or disinfectant is preferably 18 to 20 billion/g, more preferably 19 to 20 billion/g, and most preferably 20 billion/g. The bacterial agent or disinfectant of the present invention can efficiently degrade dinitroaniline pesticides, especially butralin. The disinfectant of the present invention is preferably used in vegetables and/or fruits. According to the specific embodiments of the present invention, after the bacterial agent treats the soil with high residue of butralin for 3 to 8 days, butralin is degraded into 2-{[(1Z)-2-methylcyclopropylidene]amino}-5-(2-methylprop-2-yl)-3-nitroaniline, 5-(2-methylprop-2-yl)-3-nitrobenzene-1,2-diamine, 2-{[(2E)-butyl-2-ylidene]amino}-5-(2-methylprop-2-yl)-3-nitroaniline, 2-(butyl-2-ylamino)-5-(2-methylprop-2-yl)-3-nitroaniline and other four metabolites. It can be seen that the bacterial agent provided by the present invention has a good degradation effect.

The present invention also preferably provides a method for preparing the fermentation broth of the Bacillus Velez FC02, comprising the following steps:

The activated Bacillus Velez FC02 is inoculated into a seed liquid culture medium for culturing to obtain a seed liquid of Bacillus Velez FC02;

The Bacillus Velez subtilis FC02 seed liquid is inoculated into a liquid culture medium for shaking culture to obtain a Bacillus Velez subtilis FC02 fermentation liquid.

The activation method of the present invention is preferably to inoculate Velez subtilis FC02 onto NA medium under sterile conditions to obtain activated Velez subtilis FC02. The activation time of the present invention is preferably 12 hours; the activation temperature is preferably 30-35°C; the NA medium preferably includes the following components: peptone 10g/L, beef extract powder 3g/L, sodium chloride 5g/L and agar 15-20g/L, and the pH is preferably 7.1-7.5; when performing the activation, the inoculation amount of Velez subtilis FC02 is preferably 1-3 rings.

After obtaining the activated Bacillus Velez subtilis FC02, the present invention preferably inoculates the activated Bacillus Velez subtilis FC02 into a seed liquid culture medium for cultivation to obtain Bacillus Velez subtilis FC02 seed liquid. The cultivation time of the present invention is preferably 12 to 16 hours, and the temperature is preferably 30 to 35°C; the seed liquid culture medium is preferably LB culture medium; the LB culture medium preferably includes the following components of tryptone 10g/L, yeast extract 5g/L and sodium chloride 10g/L, and the pH is preferably 7.2 to 7.5; during the cultivation, the inoculation amount of Bacillus Velez subtilis FC02 is preferably 1 to 3 rings.

After obtaining the Bacillus Velez subtilis FC02 seed solution, the present invention preferably inoculates the Bacillus Velez subtilis FC02 seed solution into a liquid culture medium for shaking culture to obtain Bacillus Velez subtilis FC02 fermentation liquid. The temperature of the shaking culture of the present invention is preferably 30-40°C, more preferably 32-38°C, and more preferably 34-36°C; the rotation speed of the shaking culture is preferably 180-200r/min; the standard for the end of the shaking culture is preferably that Bacillus Velez subtilis FC02 enters the logarithmic growth phase, that is, when the OD ₆₀₀ value of Bacillus Velez subtilis FC02 is preferably 3.0, the shaking culture is ended. During the shaking culture of the present invention, the volume ratio of the Bacillus Velez subtilis FC02 seed solution to the liquid culture medium is preferably 1-10:30-500, and more preferably 1-40.

The liquid culture medium of the present invention preferably includes the following components in percentage by weight: 2% to 10% glucose, 2% to 10% beef extract, and 1% to 5% calcium carbonate; that is, 2% to 10% glucose, 2% to 10% beef extract, and 1% to 5% calcium carbonate are mixed with 1000mL deionized water to obtain the liquid culture medium of the present invention. The initial pH of the liquid culture medium of the present invention is preferably 7.0 to 7.2; the present invention preferably uses hydrochloric acid to adjust the pH of the liquid culture medium. The liquid culture medium of the present invention is preferably a liquid culture medium obtained after sterilization at 120°C for 20 minutes.

After obtaining the fermentation liquid of Bacillus Velez subtilis FC02, the present invention preferably adjusts the concentration of the fermentation liquid of Bacillus Velez subtilis FC02; the present invention does not limit the method of adjustment, and any method known to those skilled in the art can be used. The concentration of Bacillus Velez subtilis FC02 in the fermentation liquid of Bacillus Velez subtilis FC02 obtained by the adjustment of the present invention is preferably 1×10 ⁵ to 1×10 ⁸ cfu/mL, more preferably 1×10 ⁷ to 1×10 ⁸ cfu/mL.

In the present invention, the bacterial agent or disinfectant also preferably includes an auxiliary material; the volume mass ratio of the active ingredient to the auxiliary material is preferably 200-500mL:300-600g, more preferably 250-450mL:350-550g, and most preferably 300-400mL:400-500g; in a specific embodiment of the present invention, the volume mass ratio of the active ingredient to the auxiliary material is preferably 250mL:300g. The auxiliary material of the present invention preferably includes a carrier and an auxiliary agent; the carrier preferably includes one or more of white carbon black, diatomaceous earth, kaolin, and bentonite, and more preferably includes white carbon black and/or bentonite. The carrier of the present invention can effectively adsorb Bacillus Velez FC02.

The auxiliary agent of the present invention preferably includes a wetting agent, a dispersant and a protective agent; the wetting agent preferably includes one or more of fatty alcohol polyoxyethylene ether (AEO), sucrose fatty acid ester, Tween 80, ethoxylated alkyl sodium sulfate, sodium dodecylbenzene sulfonate, and sodium lignin sulfonate, and more preferably includes sodium dodecylbenzene sulfonate and/or sodium lignin sulfonate.

The dispersant of the present invention preferably includes one or more of calcium chloride, polyvinyl alcohol, sodium tripolyphosphate (STPP), soluble starch, ammonium sulfate, and polyethylene glycol, and more preferably includes polyvinyl alcohol.

The protective agent of the present invention preferably includes one or more of dextrin, silicon dioxide, hydroxymethyl cellulose, sodium hydroxymethyl cellulose, and L-ascorbic acid, and more preferably includes dextrin and/or silicon dioxide. The protective agent of the present invention mainly has the function of water retention; dextrin in the protective agent of the present invention has the functions of slow setting, water retention, and increasing workability; silicon dioxide has the functions of anti-caking agent and shielding, achieving the purpose of anti-ultraviolet aging and heat aging, and increasing the heat insulation of the coating; hydroxymethyl cellulose has the characteristics of adhesion, thickening, flow, emulsification and dispersion, excipient, water retention, protective colloid, film forming, acid resistance, salt resistance, suspension, etc.; sodium hydroxymethyl cellulose has the functions of adhesion, thickening, enhancement, emulsification, water retention, suspension, etc.; L-ascorbic acid has high reducing property and is also an anticoagulant. After adding the protective agent, the damage to the strain during ultraviolet irradiation is avoided, and the purpose of stable release can be achieved in the environment. It is stable in storage under normal temperature and pressure.

The bacterial agent or disinfectant of the present invention preferably includes the following components in percentage by mass: 40% to 50% of active ingredients and 50% to 60% of auxiliary materials, more preferably 43% to 47% of active ingredients and 53% to 58% of auxiliary materials; the auxiliary materials include the following components in percentage by mass: 70% to 90% of carrier, 5% to 10% of wetting agent, 5% to 10% of dispersant, 2% to 4% of protective agent, more preferably 80% to 90% of carrier, 5% to 8% of wetting agent, 5% to 8% of dispersant, 2% to 3% of protective agent. The types of auxiliary materials of the present invention have been described in detail above and will not be repeated here; the present invention does not specifically limit the types and proportions of the auxiliary materials, that is, conventional combinations are sufficient, and only the percentage by mass of the bacterial agent provided by the present invention needs to be satisfied.

The present invention also provides a method for preparing the bacterial agent or disinfectant described in the above technical solution, comprising the following steps:

After the active ingredients are mixed with auxiliary materials, they are dried to obtain a bacterial agent or disinfectant.

In the present invention, the active ingredients are mixed with the auxiliary materials and then dried to obtain the bacterial agent. The active ingredients and auxiliary materials have been described in detail in the technical scheme of the bacterial agent, which will not be repeated here.

The mixing method of the present invention is preferably stirring; the stirring temperature is preferably 30-40°C, more preferably 32-38°C, and more preferably 34-36°C; the stirring speed is preferably 50-200rpm, more preferably 80-180rpm, and more preferably 100-150rpm; the end standard of the stirring is preferably that the auxiliary material becomes flocculent.

In the present invention, the drying is preferably carried out in an electric constant temperature blast drying oven; the drying is preferably constant temperature drying; the drying temperature is preferably 35 to 40°C, more preferably 40°C; the drying time is preferably 8 to 11 hours, more preferably 10 hours.

Before the active ingredient of the present invention is mixed with the auxiliary material, it is preferred that the carrier and auxiliary agent in the auxiliary material are mixed and dried before mixing with the bacterial agent. The mixed drying method of the present invention is preferably spray drying; the spray drying conditions are preferably 180°C inlet air temperature and 60°C outlet air temperature. The mixing method of the present invention can avoid mixing the bacterial agent with the auxiliary agent or carrier, which is not conducive to the survival of the strain when spray drying is performed at a temperature higher than 150°C, thereby greatly reducing the amount of bacteria.

After the drying, the present invention preferably pulverizes the dried bacterial agent obtained by drying to obtain the bacterial agent; the present invention preferably adopts an airflow pulverizer for the pulverization; the operating parameters of the airflow pulverizer during pulverization are preferably: material holding capacity 0.5kg; feed frequency 3Hz; pulverization working medium pressure 0.8MPa, classifier speed 3300r/min; induced draft fan flow rate ^15m3 /min; pulverization time 60min; the bacterial agent obtained after pulverization is preferably the undersize obtained after passing through a 400-mesh sieve.

Based on the advantages of the Bacillus Velez FC02 provided above, the present invention provides the use of the Bacillus Velez FC02 described in the above technical solution or the bacterial agent or disinfectant described in the above technical solution or the bacterial agent or disinfectant prepared by the preparation method described in the above technical solution in the degradation of dinitroaniline pesticides in an environment, and further, the use of the Bacillus Velez FC02 described in the above technical solution or the bacterial agent or disinfectant described in the above technical solution or the bacterial agent or disinfectant prepared by the preparation method described in the above technical solution in the degradation of butralin in an environment. In the present invention, the environment preferably includes soil and/or water.

The present invention also provides a method for efficiently degrading dimethylaniline pesticides, comprising the following steps:

The bacterial agent or disinfectant described in the above technical solution or the bacterial agent or disinfectant prepared by the preparation method described in the above technical solution is applied to the area contaminated by dimethylaniline pesticides. The application method of the present invention preferably includes broadcasting; the dinitroaniline pesticides of the present invention preferably include dinitroaniline pesticide herbicides, more preferably butralin. The area of the present invention preferably includes dimethylaniline pesticide high residue soil, more preferably dimethylaniline pesticide high residue soil after planting crops, more preferably butralin high residue soil after planting crops. The present invention can treat dimethylaniline pesticide high residue soil with a residue of preferably 100 to 1000 mg/kg of dimethylaniline pesticides, more preferably 100 to 500 mg/kg or 500 to 1000 mg/kg, more preferably 150 to 200 kg/kg or 800 to 1000 mg/kg. After the bacterial agent is applied, the active ingredient Bacillus Velez FC02 will be released from the bacterial agent to colonize in the soil over time, degrading butralin in the soil.

In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

Medium preparation:

Enrichment liquid medium: beef extract 5g, peptone 10g, NaCl 10g, water 1000mL, pH 7.1-7.5;

Minimal mineral salts medium (MSM): (NH ₄ ) ₂ SO ₄ 2.0 g, MgSO ₄ ·7H ₂ O 0.2 g, CaCl ₂ ·2H ₂ O0.01 g, FeSO ₄ ·7H ₂ O 0.001 g, Na ₂ HPO ₄ ·12H ₂ O 1.5 g, and KH ₂ PO ₄ 1.5 g;

NA medium: peptone 10g, beef extract powder 3g, sodium chloride 5g, agar 15-20g, distilled water 1000mL, pH 7.1-7.5;

Liquid culture medium, also known as fermentation medium: 10 g glucose, 10 g beef extract, 3 g calcium carbonate, 1000 mL deionized water, adjusted to an initial pH of 7.2 with hydrochloric acid, sterilized at 120 ° C for 20 min;

Seed liquid culture medium: tryptone 10g, yeast extract 5g, NaCl 10g, distilled water 1000mL, pH 7.1-7.5;

LB medium: 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, 1000 mL distilled water, pH 7.2-7.5.

Example 1

1. Enrichment, screening and purification of butralin-degrading strains

Take ^1cm2 cubes from the wooden board of the Southern Song Dynasty shipwreck "Nanhai No. 1", put them in a conical flask containing 100mL enrichment liquid culture medium with a concentration of 50mg/L of butadine, and culture them on a shaker at 28℃ and 200r/min for one week; then transfer them to the next batch of enrichment medium with a concentration of 100mg/L of butadine according to an inoculum of 10% of the volume of the enrichment liquid culture medium, and continue to culture for one week; then transfer them to an enrichment medium with a concentration of 200mg/L of butadine according to an inoculum of 10% of the volume of the enrichment liquid culture medium, and culture them for one week; then transfer them to an enrichment medium with a concentration of 200mg/L of butadine according to an inoculum of 10% of the volume of the enrichment liquid culture medium, and culture them for one week; then transfer them to an enrichment medium with a concentration of 200mg/L of butadine according to an inoculum of 10% of the volume of the enrichment liquid culture medium, and culture them for one week. /L of basic inorganic salt medium (MSM), and continue to culture for one week; then transfer the inoculation amount of 10% of the volume of the enriched liquid medium to the basic inorganic salt medium containing 200 mg/L of butralin, and continue to culture for one week; then take 100 μL of the basic inorganic salt medium fermentation liquid and spread it on NA solid medium plates, the spread plates are placed in a constant temperature incubator at 28°C and cultured for 2d to 3d, single colonies with different morphological characteristics are selected and inoculated on a separation medium containing 200 mg/L of butralin, and the plate streak method is used for three times of separation and purification to obtain strains I to IV.

During the initial screening of strains, a solid plate was prepared by adding butralin to NA medium. The plate was yellow after being prepared. The degradation ability of strains I to IV on butralin is shown in Figure 1, where X1 is strain I, X2 is strain II, X3 is strain III, and X5 is strain IV. The results of bacterial coating showed that strain II (upper right) had the most significant degradation ability, that is, the most yellow was eliminated in the plate; strains I, III and IV had no degradation ability or had a lower degradation ability (upper left, lower left and lower right).

2. Phenotypic characteristics and physiological and biochemical analysis of strain II

2.1 Morphological characteristics of strain II

The strain II to be tested was diluted and spread on NA medium and cultured at a constant temperature of 28°C. The external morphology, size, surface viscosity, transparency and odor of the colonies were observed every 6 hours.

The single colony morphology of strain II is shown in Figures 2 and 3. The single colony morphology is nearly round, light yellow, and opaque. The colony surface is smooth and has neat edges in the early stage of culture. In the later stage, the surface has wrinkles, slightly irregular edges, a bulge in the middle, and a cloud-like spread around.

2.2 Cell morphological characteristics

Use an inoculation loop to pick up three loops of freshly streaked strain II cells three times, wash with phosphate buffer, and centrifuge at 180 rpm for 10 minutes to collect the cells. Fix with 2.5% glutaraldehyde solution by volume, and prepare scanning electron microscopy and transmission electron microscopy samples according to the method of Li Guangyu et al. ([1] Li Guangyu, Zeng Xiang, Shao Zongze. Analysis of dissimilatory iron-reducing microorganisms and their mineralization products in the hydrothermal area of the South Atlantic Ridge [J]. Journal of Microbiology, 2019, 59(07): 1295-1306. DOI: 10.13343/j.cnki.wsxb.20180385.). Observe its morphology under scanning electron microscopy and transmission electron microscopy.

The colonies of strain II cultured on NA plates for about 48 hours were used as research materials. The microstructure of strain II was observed under a transmission electron microscope. The hyphae cell wall and cell membrane of strain II were closely connected and the structure was complete. The cell matrix was evenly distributed, the cross section was regular and the structure was clear. The nucleus and other organelles were in a good growth state. Observed under a scanning electron microscope, the results are shown in Figures 4 and 5. Strain II was rod-shaped, full, with blunt rounded ends, no capsule, and a size of (1.6-2.2)×(0.68-0.70) microns.

2.3 Physiological and biochemical identification tests

Strain II was diluted and spread on NA medium and cultured at 28°C to observe the external morphology, size, surface viscosity, transparency and smell of the colonies. The physiological and biochemical characteristics of strain II were identified according to the methods of the "Common Bacteria System Identification Manual" and the "Berger's Bacteria Identification Manual". Strain II can also be called antagonistic strain II. The identification results are shown in Table 1.

Table 1 Physiological and biochemical characteristics identification results

The physiological and biochemical characteristics of strain II are shown in Table 1. In the methyl red test, the color of the culture medium containing strain II did not change significantly after adding methyl red indicator, which was a negative reaction; the color of the culture medium containing solution A and solution B did not change significantly after adding solution A and solution B, which was a negative reaction; strain II can grow normally in 0.1% Congo red medium and anaerobic medium; it has no motility; the culture medium did not change significantly after growing in citrate medium for 3 days, so citrate cannot be used; a transparent circle formed around the colony of starch medium with iodine solution added, which was a positive reaction; the gelatin liquefaction results showed that the gelatin clot did not melt, which was a negative reaction. Therefore, the strain was preliminarily identified as Bacillus through the above colony morphological characteristics and physiological and biochemical characteristics.

3. Construction of the phylogenetic tree of strain II

The genome of strain II to be tested was extracted, and PCR amplification was performed with primers 27F and 1492R to obtain the 16SrDNA sequence of strain II, wherein 27F is specifically as shown in SEQ ID NO: 2: 5'-AGAGTTTGATCCTGGCTCAG-3', and 1492R is specifically as shown in SEQ ID NO: 3: 5'-CTACGGCTACCTTGTTACGA-3';

The PCR product was recovered using the AxyPrep DNA gel recovery kit, and the specific operation was carried out according to the kit instructions. After the PCR amplification product was detected by 1% agarose gel electrophoresis, the gel block where the target band was located was cut and sequenced. The sequencing result of the 16S rDNA sequence is shown in SEQ ID No: 1: 5'-aggttacctcaccgacttcgggtgttacaaactctcgtggtgtgacgggcggtgtgtacaaggcccgggaacgtattcaccgcggcatgctgatccgcgattactagcgattccagcttcacgcagtcgagttgcagactgcgatccgaactgagaacagatttgtgggattggcttaacctcgcggtttcgctgccctttgttctgtccattgtagcacgtgtgtagcccaggtcataaggggcatgatgatttgacgtcatccccaccttcctccggtttgtcaccggcagtcaccttagagtgcccaactgaatgctggcaactaagatcaagggttgcgctcgttgcg ggacttaacccaacatctcacgacacgagctgacgacaaccatgcaccacctgtcactctgcccccgaaggggacgtcctatctctaggattgtcagaggatgtcaagacctggtaaggttcttcgcgttgcttcgaattaaaccacatgctccaccgcttgtgcgggcccccgtcaattcctttgagtttcagtcttgcgaccgtactccccaggcggagtgcttaatgcgttagctgcagcactaaggggcggaaaccccctaacacttagcactcatcgtttacggcgtggactaccagggtatctaatcctgttcgctccccacgctttcgctcctcagcgtcagttacagaccagag agtcgccttcgccactggtgttcctccacatctctacgcatttcaccgctacacgtggaattccactctcctcttctgcactcaagttccccagtttccaatgaccctccccggttgagccgggggctttcacatcagacttaagaaaccgcctgcgagccctttacgcccaataattccggacaacgcttgccacctacgtattaccgcggctgctggcacgtagttagccgtggctttctggttaggtaccgtcaaggtgccgccctatttgaacggcacttgttcttccctaacaacagagctttacgatccgaaaaccttcatcactcacgcggcgttgctccgtcagactttcgtcc attgcggaagattccctactgctgcctcccgtaggagtctgggccgtgtctcagtcccagtgtggccgatcaccctctcaggtcggctacgcatcgtcgccttggtgagccgttacctcaccaactagctaatgcgccgcgggtccatctgtaagtggtagccgaagccaccttttatgtctgaaccatgcggttcagacaaccatccggtattagccccggtttcccggagttatcccagtcttacaggcaggttacccacgtgttactcacccgtccgccgctaacatcagggagcaagctcccatctgtccgctcgacttgcatgtattaggcacgccgccagcgttcgtcctga-3'.

The spliced sequence files were compared with the data in the NCBI 16S database using the NCBI Blast program to obtain the species information with the greatest sequence similarity to the species to be tested. The Neighbor-Joining method of MEGA6.0 software was used to construct a phylogenetic tree, as shown in Figure 6. FC02 in Figure 6 is strain II.

The 16S rDNA sequence of strain II was extracted and amplified by agarose gel electrophoresis. The DNA band was single and bright. Compared with the results of Trans2KPlusII DNA marker electrophoresis, the band of strain II was between 1000 and 2000 bp. The sequence length was 1,437 bp. After comparison with the GenBank database, the results showed that the similarity between strain II and the model strain Bacillus velezensis CR-502 (T) (AY603658) was 99%. Combined with the phylogenetic tree, strain II was identified as Bacillus velezensis and named Bacillus velezensis FC02.

Example 2

Degradation of butralin by Bacillus velez FC02

The Velez Bacillus FC02 strain is cultured by a spore-forming fermentation method, that is, the Velez Bacillus FC02 strain is cultured by the method of step 1), and the degradation effect on butralin is determined, and the steps are as follows:

1) Cultivation method of Bacillus Velez FC02 fermentation liquid: under sterile conditions, inoculate Bacillus Velez FC02 onto NA medium with an inoculation loop for activation for 12 hours, pick Bacillus Velez FC02 with an inoculation loop and place it in a 250mL triangular flask containing 50mL seed liquid medium, pick 3 loops, and culture at 30°C for 15 hours to obtain Bacillus Velez FC02 seed liquid; inoculate Bacillus Velez FC02 seed liquid into liquid culture medium, the volume ratio of Bacillus Velez FC02 seed liquid to liquid culture medium is 1:40, and after shaking culture at 35°C and 200r/min for 16 hours, detect the concentration of the bacterial liquid. When the bacterial liquid content exceeds 1×10 ⁸ cfu/mL, it is Bacillus Velez FC02 fermentation liquid;

2) Inoculation and cultivation, inoculating the fermentation liquid of Bacillus Velez FC02 into fresh MSM, the volume ratio of Bacillus Velez FC02 seed liquid to fresh MSM is 1:40, adjusting the bacterial concentration to make its _OD600 be 1.0, and then adding butralin to the fresh MSM to make its final concentration 200 mg/L to obtain a degradation bacterial liquid;

The prepared degradation bacterial solution was placed on a constant temperature shaker at 30°C and 200 r/min for 6 days. At the same time, fresh MSM containing butralin without inoculation was used as a control. The final concentration of butralin in fresh MSM containing butralin was 200 mg/L, recorded as CK, and each treatment was repeated 3 times.

3) Butylamine extraction method: After the culture is completed, directly draw 2 mL of the fermentation liquid of Bacillus Velez FC02, add 10 mL of ethyl acetate, vortex at 2000 r/min for 10 min, and centrifuge at 4000 r/min for 10 min. The solution is layered, and 2 mL of the supernatant is drawn, concentrated to dryness on a nitrogen blower at 40°C, and the volume is adjusted to 2 mL with methanol. After filtering through a 0.22 μm organic phase filter membrane, the residual amount of butylamine is determined.

The degradation color of butralin is shown in Figure 7, where the left side is the control and the right side is the degradation bacterial solution. When butralin is dissolved in the basic inorganic salt culture medium at a concentration of 200 mg/L, it is a yellow liquid in the culture medium. After being metabolized by Bacillus Velez FC02, a dark red substance appears as the metabolite, indicating that Bacillus Velez FC02 can effectively metabolize butralin.

Liquid chromatography was used to determine the changes in the content of butaline and the content of its metabolites, wherein the liquid chromatography determination conditions of butaline were as follows: Waters e2695 high performance liquid chromatograph (with 2489 UV/visible detector); chromatographic column: Waters XBridge C ₁₈ (5 μm, 4.6 mm×250 mm); mobile phase B was chromatographically pure acetonitrile, mobile phase C was pure water, mobile phases B and C were gradient eluted, gradient elution conditions: 0-6 min, 50% acetonitrile; 6-21 min, 80% acetonitrile; flow rate was 1.0 mL/min. The injection volume was 10 μL; the detection wavelength was 240 nm; butaline retention time: 11.8 min.

The liquid chromatograms are shown in Figures 10 to 13, wherein A is 2-{[(1Z)-2-methylcyclopropylidene]amino}-5-(2-methylprop-2-yl)-3-nitroaniline, B is 5-(2-methylprop-2-yl)-3-nitrobenzene-1,2-diamine, C is 2-{[(2E)-butyl-2-ylidene]amino}-5-(2-methylprop-2-yl)-3-nitroaniline, and D is 2-(butyl-2-ylamino)-5-(2-methylprop-2-yl)-3-nitroaniline. It can be seen from Figures 10 to 13 that the present invention After the provided bacterial agent was used to treat butralin for 6 days, butralin was degraded into four metabolites, namely, 2-{[(1Z)-2-methylcyclopropylidene]amino}-5-(2-methylprop-2-yl)-3-nitroaniline, 5-(2-methylprop-2-yl)-3-nitrobenzene-1,2-diamine, 2-{[(2E)-butyl-2-ylidene]amino}-5-(2-methylprop-2-yl)-3-nitroaniline, and 2-(butyl-2-ylamino)-5-(2-methylprop-2-yl)-3-nitroaniline. The bacterial agent had a good degradation effect.

The degradation of butralin in Bacillus Velez FC02 and the growth of the strain are shown in Table 2; the degradation rate can be obtained by detecting the residual amount of butralin, and the results are shown in Figure 8, where the degradation rate = (butralin residual amount in the control culture medium - butralin residual amount in the treatment culture medium) / butralin residual amount in the control culture medium × 100%.

Table 2 Degradation of butaline in Bacillus velez FC02 and growth of strains

As shown in Table 2 and Figure 8, the growth of the strain slowly decreased with the extension of time. The concentration of butadine increased with the rate of change of the strain, and the content decreased significantly. The residual amount of butadine was 37.68 mg/L at 3d (the degradation rate reached 81.16%). With the large reduction of the number of colonies, the degradation of butadine also tended to be flat. It may be that as the pesticide is degraded, the carbon and nitrogen sources available for use decrease, the growth rate of the strain decreases, and the degradation of butadine also slows down. Although the content of butadine in CK decreases slowly, the decline ratio is much lower than the treatment of inoculating degradation bacteria liquid.

Example 3

Single Factor Test on Degradation of Butralin by Bacillus Velez FC02

1) Effect of inoculum size on the degradation characteristics of butralin

A single colony of freshly cultured strain FC02 was picked and inoculated into 100 mL LB medium. The culture was cultured at 28°C and 200 r/min until the late logarithmic phase. The cells were collected by centrifugation at 5000 r/min for 5 min, washed three times with fresh basal inorganic salt medium (MSM), and then resuspended with the same medium.

The strain FC02 seed liquid was inoculated into fresh inorganic salt medium (MSM) at different temperatures, and the bacterial concentration was adjusted to _0.50 , 1.0, 1.5, 2.0, 2.5, and 3.0, respectively, and then butralin was added to the MSM to make the final concentration 100 mg/L. The culture was carried out in a shaking table at 28°C and 200 r/min, and samples were taken for determination after 3 days. At the same time, MSM without bacteria inoculation was set as a control, and 3 parallel experiments were performed for each sample treatment. The detection method was the same as in Example 2.

2) Effect of temperature on the degradation characteristics of butralin

Culture the bacteria in the manner of 1). Inoculate the strain FC02 seed solution into fresh inorganic salt medium (MSM) at different temperatures, adjust the bacterial concentration to make its OD ₆₀₀ 1.0, and then add butralin to the MSM to make its final concentration 100 mg/L. Culture in a shaking incubator at 200 r/min at different initial temperatures of 20, 25, 30, 35, 40 and 45°C, take samples for determination after 3 days, and set the culture solution without bacteria as a control. Each sample is processed for 3 parallel experiments. The detection method is the same as in Example 2.

3) Effect of pH on the degradation characteristics of butralin

Culture the bacteria in the manner of 1). Inoculate the strain FC02 seed liquid into fresh inorganic salt medium (MSM) of different pH, adjust the bacterial concentration to make its OD ₆₀₀ be 1.0, and then add butyric acid to MSM to make its final concentration be 100 mg/L. The initial pH of the inorganic salt medium (MSM) is set to 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0, respectively, and the pH is adjusted with NaOH and HCl aqueous solutions. Culture in a shaking table at 28°C and 200r/min, and samples are taken for determination after 3 days. At the same time, the culture solution without bacteria inoculation is set as a control, and 3 parallel experiments are performed for each sample treatment. The detection method is the same as in Example 2.

The degradation results of butralin by strain FC02 under different inoculation conditions are shown in Table 3 and Figure 14.

Table 3 The results of the degradation test of butralin by strain FC02 under different inoculation conditions

The results are shown in Table 3 and Figure 14. The inoculation amount of strain FC02 was adjusted to make the cell concentration gradient in the degradation bacterial solution 0.5-3.0, and the effect of the initial concentration of the strain on the degradation of butaline was detected and analyzed. As the inoculation amount of strain FC02 increased, the degradation rate of butaline gradually accelerated. When the inoculation OD ₆₀₀ was 0.5, the degradation rate of butaline by strain FC02 in 24 hours was 23.50%. When the cell inoculation amount of the strain OD ₆₀₀ was 2-3, strain FC02 could degrade more than 90% of butaline in 24 hours. The degradation rate of butaline was positively correlated with the inoculation amount of strain FC02.

The degradation results of butralin by strain FC02 under different temperature conditions are shown in Table 4 and Figure 15.

Table 4 The results of the degradation test of butralin by strain FC02 at different temperatures

The results are shown in Table 4 and Figure 15. When the temperature is 30-40°C, the strain FC02 has a better degradation effect on butralin, with a degradation rate of >90%. This may be because this temperature range is more suitable for the growth of the strain. At 35°C, the degradation rate was 97.05%, reaching the best.

The results of the experiment on the degradation of butralin by strain FC02 under different pH conditions are shown in Table 5 and Figure 16.

Table 5 Degradation test results of butralin by strain FC02 under different pH conditions

The results are shown in Table 5 and Figure 16. The degradation rate of butralin by strain FC02 is different. At pH 7.0-9.0, the degradation rate is higher, exceeding 90%; at pH <5, the amount of butralin degraded by strain FC02 is less than 60%; at pH 8.0, the degradation rate reaches the highest, which is 96.00%.

Example 4

Degradation of butralin in soil by Bacillus velez FC02

The steps of preparing a bacterial agent containing Bacillus Velez FC02 and determining the degradation effect of butralin in soil are as follows:

1) Preparation of soil samples

Soil samples that have not been fertilized with butralin within three years were collected, naturally air-dried, and sieved to obtain fine soil particles with a diameter of 2 mm. This embodiment uses two types of soil samples, sterilized and unsterilized, and the sterilized soil samples are sterilized at 121°C for 60 minutes;

Add butralin to the sterilized and non-sterilized soil samples respectively until the concentration of butralin in the soil sample reaches 200 mg/kg, stir evenly, and record the sterile soil sample containing butralin as soil sample I and the non-sterile soil sample containing butralin as soil sample II;

Preparation method of soil sample with 200 mg/kg of butralin concentration: 2.67 mL of 15000 mg/L butralin stock solution was added to 200 g of soil. Sterile deionized water was added appropriately until the humidity reached 10%. The test was carried out in a sterilized plastic container.

2) Preparation of a bacterial agent containing Bacillus Velezii FC02, hereinafter referred to as a bacterial agent

2.1) Under sterile conditions, inoculate Bacillus Velez FC02 onto NA medium with an inoculation loop for activation for 12 hours, pick Bacillus Velez FC02 with an inoculation loop and place it in a 250mL Erlenmeyer flask containing 50mL seed liquid culture medium, pick 3 loops, and culture at 30°C for 15 hours to obtain Bacillus Velez FC02 seed liquid; inoculate Bacillus Velez FC02 seed liquid into 400mL liquid culture medium, at an inoculation amount, the volume ratio of Bacillus Velez FC02 seed liquid to liquid culture medium is 1:40, and after shaking culture at 35°C and 200r/min for 16 hours, detect the bacterial liquid concentration. When the bacterial liquid content exceeds 1×10 ⁸ cfu/mL, it is the fermentation liquid of Bacillus Velez FC02;

2.2) Synthesis of auxiliary materials: 85wt.% white carbon black, 6wt.% sodium lignin sulfonate, 6wt.% polyvinyl alcohol and 3wt.% sodium hydroxymethyl cellulose were mixed evenly and spray-dried at an inlet air temperature of 180°C and an outlet air temperature of 60°C. The prepared carrier powder was sieved through a 400-mesh sieve to prepare an auxiliary material for Bacillus Velez FC02.

2.3) 250 mL of the fermentation liquid of Bacillus Velezii FC02 was slowly added to 300 g of the auxiliary material, and stirred at 30°C and 150 r/min until the auxiliary material became flocculent. After being dried in an electric constant temperature blast drying oven at 40°C for 10 h, the auxiliary material was crushed by an air flow mill. The operating parameters of the air flow mill were as follows: material holding capacity 0.5 kg; feeding frequency 3 Hz; crushing working fluid pressure 0.8 MPa, classifier speed 3300 r/min; induced draft fan flow rate 15 m ³ /min; crushing time 60 min, and the auxiliary material was passed through a 400-mesh sieve. The bacterial agent was vacuum-packaged to obtain a bacterial agent that efficiently degrades butralin.

The spore content, suspension rate, wetting time and moisture content in the microbial agent were detected. The detection method of the spore content was as follows: 1g of sample was diluted 1 times, 100 times, 1000 times, 10000 times... and then plated to count the colonies. The suspension rate was determined according to GB/T 14825-2006 "Determination of Suspension Rate of Pesticides", the wetting time was determined according to GB/T 5451-2001 "Determination of Wettability of Pesticides", and the moisture content was determined according to "Standard Determination of Moisture in Pesticides".

The test results showed that the spore content was 20 billion/g, the suspension rate was 90%, the wetting time was 59s, and the moisture content was 3.49%; the test results showed that the suspension performance of the bacterial agent was excellent, the wetting time was far lower than the value specified by the corresponding national standards, and it showed a good wetting effect.

3) Inoculation of bacterial agents

Take 5 g of the bacterial agent, dilute it 2 times with sterile water, and then mix it appropriately with 200 g of soil sample I in step 1). Similarly, take 5 g of the bacterial agent, dilute it 2 times with sterile water, and then mix it appropriately with 200 g of soil sample II in step 1), and record them as experimental group 1 and experimental group 2, respectively. Soil sample I and soil sample II that were not inoculated with the bacterial agent were used as controls, and were recorded as control group 1 and control group 2, respectively.

4) Sampling and extraction

10 g of the above soil samples were collected on the 0th, 2nd, 4th, 6th, 8th and 10th day after inoculation for analysis of butralin residues. The specific method was as follows: ultrasonically extract the sample with 20 mL of acetonitrile for 15 min, shake it for 10 min, centrifuge it at 4000 r/min for 10 min, and take the supernatant; take 1 mL of the supernatant and filter it through a 0.22 μm organic phase filter for high performance liquid chromatography analysis.

The degradation rate of the fungicide butarin in soil is shown in Figure 9 and Table 6. A in Figure 9 is the control group 1 (butarin + non-sterilized), B is the experimental group 1 (butarin + fungicide + sterilized soil), C is the control group 2 (butarin + sterilized soil), and D is the experimental group 2 (butarin + fungicide + non-sterilized soil).

Table 6 Degradation of butralin pesticide in soil by Bacillus Velez FC02 (mg/L)

The test results recorded in Figure 9 and Table 6 show that in sterilized soil, compared with the control group 1 without adding the microbial agent, the removal rate of butarine in the experimental group 1 after adding the microbial agent was significantly improved. In non-sterile soil, compared with the control group 2 without adding the microbial agent, the removal rate of butarine in the experimental group 2 after adding the microbial agent was significantly improved. In general, the microbial agent provided by the present invention has a significant degradation effect on butarine in soil, and the degradation rate is increased by more than 50% compared with the soil without adding the microbial agent. It shows that Bacillus Velez FC02 has the potential to repair soil, which is the first exploration of the bioremediation potential of butarine-degrading microbial agents in soil.

In summary, the strain of the present invention can effectively degrade dinitroaniline pesticide residues, especially butralin residues, in a short time, protect the ecological environment and human health, and is easy to use, low in cost, and has a removal rate of more than 80%. It can be used to repair water bodies, soil, etc. contaminated by dinitroaniline pesticides, degrade dinitroaniline pesticide residues on crops, solve the problem of excessive pesticide residues and environmental pollution in agricultural production, and protect the safety of crop production and human health.

Although the present invention has been disclosed as above in the form of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims (9)

1. Bacillus bailii (Bacillus velezensis) FC02, wherein the preservation number of the Bacillus bailii FC02 is CGMCC No.22512.

2. A microbial inoculum or disinfectant for degrading dinitroaniline pesticides, characterized in that the active ingredient of the microbial inoculum or disinfectant comprises the fermentation broth, seed liquid or thallus of bacillus bailii FC02 according to claim 1.

3. A microbial agent or disinfectant according to claim 2, wherein the effective viable count in the microbial agent or disinfectant is 180 to 200 hundred million/g.

4. A microbial agent or disinfectant according to claim 2, wherein the microbial agent or disinfectant further comprises an adjunct; the auxiliary materials comprise a carrier and an auxiliary agent.

5. The microbial inoculum or disinfectant according to claim 4, wherein the volume/mass ratio of the active ingredient to the auxiliary material is 200-500 ml/300-600 g.

6. The method for preparing the microbial inoculum or the disinfectant according to any one of claims 3-5, which is characterized by comprising the following steps:

Mixing the effective components with adjuvants, and oven drying to obtain microbial inoculum or disinfectant.

7. The method according to claim 6, wherein the drying is constant temperature drying, the temperature of the drying is 35-40 ℃ and the time is 8-11 h.

8. The bacillus belicus FC02 as claimed in claim 1 or the microbial inoculum or disinfectant as claimed in any one of claims 2 to 5 or the use of the microbial inoculum or disinfectant prepared by the preparation method as claimed in claim 6 or 7 in a degrading environment.

9. The method for efficiently degrading the butralin is characterized by comprising the following steps of:

Applying the microbial inoculum or disinfectant according to any one of claims 2-5 or prepared by the preparation method according to claim 6 or 7 to a butralin contaminated area.