Microbial preparation for degrading carbendazim and preparation method and application thereof

Abstract

The invention discloses a microbial preparation for degrading carbendazim as well as a preparation method and application thereof, and relates to the technical field of biological preparations. The microbial preparation comprises mixed bacteria, the mixed bacteria comprise sphingomonas, burkholderia cepacia, bacillus amyloliquefaciens, bacillus megatherium, trichoderma koningii, streptomyces, azospirillum brazilianum and pseudomonas fluorescens. The carbendazim can be effectively and rapidly degraded, the growth of edible fungi such as termitomyces albuminosus and plants in carbendazim residual soil is improved, the microbial diversity of the soil is improved, and the bacterial community structure is enriched.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a microbial preparation for degrading carbendazim, its preparation method, and its application. Background Technology

[0002] Carbendazim, also known as benzimidazole or benzimidazole 44, is a broad-spectrum fungicide that interferes with nucleic acid synthesis and cell mitosis, effectively controlling various crop diseases caused by fungi. It is a widely used pesticide in agriculture. While its use can control plant diseases and effectively improve production efficiency, its improper use has led to serious problems such as excessive pesticide residues in agricultural products and environmental pollution. Carbendazim residues in soil and other environments inhibit microbial growth, disrupt the balance and function of ecosystems, and can also cause embryonic, reproductive, developmental, and hematological toxicity in animals. Microbial degradation of pesticide residues has advantages such as low cost and environmental friendliness. Therefore, providing a microbial preparation that can effectively degrade carbendazim is crucial. Summary of the Invention

[0003] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes a microbial preparation.

[0004] The present invention also provides a method for preparing the above-mentioned microbial preparation.

[0005] The present invention also provides a soil remediation agent comprising the above-mentioned microbial preparation.

[0006] The present invention also provides the application of the above-mentioned microbial preparation, the microbial preparation obtained by the above-mentioned preparation method, or the above-mentioned soil remediation agent.

[0007] The present invention also provides a method for degrading carbendazim.

[0008] A microbial preparation according to a first aspect of the present invention includes a mixed bacterial strain; said mixed bacterial strain includes Sphingomonas, Burkholderia cepacia, Bacillus amyloliquefaciens, Bacillus megaterium, Trichoderma cornutica, Streptomyces, Azotobacter brasiliensis, and Pseudomonas fluorescens.

[0009] The microbial preparations according to embodiments of the present invention have at least the following beneficial effects: The microbial preparations described in this embodiment have multiple functions, including synergistic degradation of carbendazim, improvement of soil physicochemical properties, and inhibition of pathogens. They can significantly improve the degradation efficiency of organic pesticide residues such as carbendazim, improve the growth of edible fungi such as black-skinned chicken mushroom and plants in soil with carbendazim residues, increase soil microbial diversity, and enrich bacterial community structure.

[0010] According to some embodiments of the present invention, the strain number of the sphingosine monocytogenes is CICC 10472.

[0011] According to some embodiments of the present invention, the strain number of Burkholderia cepacia is ATCC 25416.

[0012] According to some embodiments of the present invention, the strain of Bacillus amyloliquefaciens is numbered DSM 23117.

[0013] According to some embodiments of the present invention, the strain number of the Bacillus megaterium is CICC 10044.

[0014] According to some embodiments of the present invention, the strain of *Trichoderma cornuta* is numbered CGMCC 3.11531.

[0015] According to some embodiments of the present invention, the strain of Streptomyces is designated ATCC 25421.

[0016] According to some embodiments of the present invention, the strain of *Azotobacter brasiliensis* is designated as DSM 1690.

[0017] According to some embodiments of the present invention, the strain number of the fluorescent Pseudomonas is CICC 23876.

[0018] According to some embodiments of the present invention, the ratio of Sphingomonas, Burkholderia cepacia, Bacillus amyloliquefaciens, Bacillus megaterium, Trichoderma cornigans, Streptomyces, Azotobacter brasiliensis, and Pseudomonas fluorescens, based on CFU, is 1:(0.9-3.1):(0.9-2.1):(0.9-1.1):(0.4-1.1):(0.9-1.1):(0.9-3.1):(0.7-1.1).

[0019] According to some embodiments of the present invention, the ratio of Sphingomonas, Burkholderia cepacia, Bacillus amyloliquefaciens, Bacillus megaterium, Trichoderma cornigans, Streptomyces, Azotobacter brasiliensis, and Pseudomonas fluorescens, based on CFU, is 1:(0.9-1.1):(0.9-1.1):(0.9-1.1):(0.9-1.1):(0.9-1.1):(0.9-1.1):(0.9-1.1).

[0020] According to some embodiments of the present invention, the ratio of Sphingomonas, Burkholderia cepacia, Bacillus amyloliquefaciens, Bacillus megaterium, Trichoderma cornigeri, Streptomyces, Azotobacter brasiliensis, and Pseudomonas fluorescens, calculated in CFU, is 1:1:1:1:1:1:1:1:1:1.

[0021] According to some embodiments of the present invention, the microbial preparation has a core-shell structure, wherein the core is a calcium alginate ball encapsulating the mixed bacteria; and the shell is a chitosan layer. Both calcium alginate and chitosan are biodegradable substances, avoiding secondary pollution to the environment; simultaneously, they can slowly release the encapsulated microorganisms, avoiding adverse effects on microbial activity caused by high concentrations of carbendazim, thus improving the environmental adaptability of the microbial preparation.

[0022] According to some embodiments of the present invention, the chitosan layer is encapsulated with a carbendazim molecularly imprinted polymer. This enables specific and efficient adsorption of carbendazim, effectively improving the degradation efficiency of carbendazim.

[0023] According to some embodiments of the present invention, the preparation method of the carbendazim molecularly imprinted polymer includes the following steps: A first mixture of carbendazim, functional monomers, and acetonitrile undergoes a prepolymerization reaction to obtain a prepolymerized liquid; the prepolymerized liquid and a second mixture of crosslinking agent are subjected to deoxygenation treatment and ultrasonic treatment; under an inert gas environment, a third mixture of the second mixture and an initiator undergoes a polymerization reaction, and a solid phase is obtained by solid-liquid separation, washing, and drying to obtain the carbendazim molecularly imprinted polymer.

[0024] According to some embodiments of the present invention, the prepolymerization temperature is 0°C-4°C. For example, it can be 0°C, 1°C, 2°C, 3°C or 4°C.

[0025] According to some embodiments of the present invention, the prepolymerization time is 8 min to 20 min. For example, it can be 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min or 20 min.

[0026] According to some embodiments of the present invention, the ratio of carbendazim, functional monomer, and acetonitrile is 1 mol: (3 mol-5 mol): (30 L-40 L).

[0027] According to some embodiments of the present invention, the ratio of carbendazim, functional monomer, and acetonitrile is 1 mol : (3.9 mol-5.1 mol) : (35 L-36 L).

[0028] According to some embodiments of the present invention, the functional monomer includes methacrylic acid.

[0029] According to some embodiments of the present invention, the deoxygenation treatment includes nitrogen blowing.

[0030] According to some embodiments of the present invention, the nitrogen blowing treatment time is 5 min to 20 min. For example, it can be 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min or 20 min.

[0031] According to some embodiments of the present invention, the duration of the ultrasonic treatment is 2 min to 10 min. For example, it can be 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, or 10 min.

[0032] According to some embodiments of the present invention, the ultrasonic frequency of the ultrasonic treatment is 20 W-70 W. For example, it can be 20 W, 25 W, 30 W, 35 W, 40 W, 45 W, 50 W, 55 W, 60 W, 65 W or 70 W.

[0033] According to some embodiments of the present invention, the crosslinking agent includes ethylene glycol dimethacrylate.

[0034] According to some embodiments of the present invention, the molar ratio of the functional monomer to the crosslinking agent is 1:(4-6).

[0035] According to some embodiments of the present invention, the molar ratio of the functional monomer to the crosslinking agent is 1:(4.9-5.1).

[0036] According to some embodiments of the present invention, the inert gas includes at least one of nitrogen and argon.

[0037] According to some embodiments of the present invention, the polymerization reaction temperature is 55℃-75℃. For example, it can be 55℃, 56℃, 57℃, 58℃, 59℃, 60℃, 61℃, 62℃, 63℃, 64℃, 65℃, 66℃, 67℃, 68℃, 69℃, 70℃, 71℃, 72℃, 73℃, 74℃, or 75℃.

[0038] According to some embodiments of the present invention, the polymerization reaction time is 18 h to 48 h. For example, it can be 18 h, 20 h, 22 h, 24 h, 26 h, 28 h, 30 h, 32 h, 34 h, 36 h, 38 h, 40 h, 42 h, 44 h, 46 h, or 48 h.

[0039] According to some embodiments of the present invention, the initiator comprises azobisisobutyronitrile.

[0040] According to some embodiments of the present invention, the molar ratio of the functional monomer to the initiator is 1:(0.35-0.4).

[0041] According to some embodiments of the present invention, the molar ratio of the functional monomer to the initiator is 1:(0.36-0.38).

[0042] According to some embodiments of the present invention, the washing includes: washing with a washing solution by Soxhlet extraction until carbendazim is undetectable in the supernatant; followed by washing with methanol; the washing solution includes methanol and acetic acid in a volume ratio of (8-10):1. For example, it can be 8:1, 8.5:1, 9:1, 9.5:1, or 10:1.

[0043] The method for preparing the microbial preparation according to the first aspect embodiment of the second aspect of the present invention includes the following steps: A fourth mixture of the bacterial suspension of the mixed bacteria and sodium alginate aqueous solution is added dropwise to a calcium salt aqueous solution, cross-linked and solidified, washed, and a core is obtained; the core is immersed in a coating solution and reacted to obtain the microbial preparation. The coating solution includes carbendazim molecularly imprinted polymer and chitosan.

[0044] The preparation method according to embodiments of the present invention has at least the following beneficial effects: The preparation method in the examples uses readily available raw materials, involves simple steps, and requires minimal technical expertise.

[0045] Therefore, it is possible to prepare microbial agents that can rapidly degrade carbendazim, improve the growth of edible fungi such as black-skinned chicken mushroom and plants in soil with carbendazim residues, enhance soil microbial diversity, and enrich bacterial community structure.

[0046] According to some embodiments of the present invention, the ratio of the mixed bacteria to sodium alginate is 1×10⁻⁶. 9 CFU: (20 mg-40 mg). For example: 1×10⁻⁶ mg can be used. 9 CFU: 20 mg, 1×10 9 CFU: 22 mg, 1×10 9 CFU: 24 mg, 1×10 9 CFU: 26 mg, 1×10 9 CFU: 28 mg, 1×10 9 CFU: 30 mg, 1×10 9 CFU: 32 mg, 1×10 9 CFU: 34mg, 1×10 9 CFU: 36 mg, 1×10 9CFU: 38 mg, 1×10 9 CFU: 40 mg.

[0047] According to some embodiments of the present invention, the concentration of sodium alginate in the fourth mixture is 1%-2% (w / v)%. For example, it can be 1% (w / v)%, 1.1% (w / v)%, 1.2% (w / v)%, 1.3% (w / v)%, 1.4% (w / v)%, 1.5% (w / v)%, 1.6% (w / v)%, 1.7% (w / v)%, 1.8% (w / v)%, 1.9% (w / v)%, or 2% (w / v)%.

[0048] According to some embodiments of the present invention, the calcium salt in the calcium salt aqueous solution includes at least one of calcium chloride, calcium nitrate, and calcium sulfate.

[0049] According to some embodiments of the present invention, the calcium salt concentration in the calcium salt aqueous solution is 0.5%-5% (w / w)%. For example, it can be 0.5% (w / w)%, 1% (w / w)%, 1.5% (w / w)%, 2% (w / w)%, 2.5% (w / w)%, 3% (w / w)%, 3.5% (w / w)%, 4% (w / w)%, 4.5% (w / w)%, or 5% (w / w)%.

[0050] According to some embodiments of the present invention, the crosslinking curing temperature is 20℃-30℃. For example, it can be 20℃, 21℃, 22℃, 23℃, 24℃, 25℃, 26℃, 27℃, 28℃, 29℃ or 30℃.

[0051] According to some embodiments of the present invention, the crosslinking curing time is 20 min to 40 min. For example, it can be 20 min, 21 min, 22 min, 23 min, 24 min, 25 min, 26 min, 27 min, 28 min, 29 min, 30 min, 31 min, 32 min, 33 min, 34 min, 35 min, 36 min, 37 min, 38 min, 39 min, or 40 min.

[0052] According to some embodiments of the present invention, the dripping flow rate is 1 mL / min to 3 mL / min. For example, it can be 1 mL / min, 1.2 mL / min, 1.4 mL / min, 1.6 mL / min, 1.8 mL / min, 2 mL / min, 2.2 mL / min, 2.4 mL / min, 2.6 mL / min, 2.8 mL / min or 3 mL / min.

[0053] According to some embodiments of the present invention, the particle size of the nucleus is 1 mm to 3 mm. For example, it can be 1 mm, 1.2 mm, 1.4 mm, 1.6 mm, 1.8 mm, 2 mm, 2.2 mm, 2.4 mm, 2.6 mm, 2.8 mm or 3 mm.

[0054] According to some embodiments of the present invention, the volume-to-mass ratio of the coating solution to the carbendazim molecularly imprinted polymer is 1 mL:(10 mg-30 mg). For example, it can be 1 mL: 10 mg, 1 mL: 12 mg, 1 mL: 14 mg, 1 mL: 16 mg, 1 mL: 18 mg, 1 mL: 20 mg, 1 mL: 22 mg, 1 mL: 24 mg, 1 mL: 26 mg, 1 mL: 28 mg, or 1 mL: 30 mg.

[0055] According to some embodiments of the present invention, the chitosan concentration in the coating solution is 0.3 (w / v)%-0.8 (w / v)%. For example, it can be 0.3 (w / v)%, 0.4 (w / v)%, 0.5 (w / v)%, 0.6 (w / v)%, 0.7 (w / v)% or 0.8 (w / v)%.

[0056] According to some embodiments of the present invention, the coating solution further comprises 0.5 (v / v)%-2 (v / v)% acetic acid. For example, the concentration of the acetic acid may be 0.5 (v / v)%, 0.6 (v / v)%, 0.7 (v / v)%, 0.8 (v / v)%, 0.9 (v / v)%, 1 (v / v)%, 1.1 (v / v)%, 1.2 (v / v)%, 1.3 (v / v)%, 1.4 (v / v)%, 1.5 (v / v)%, 1.6 (v / v)%, 1.7 (v / v)%, 1.8 (v / v)%, 1.9 (v / v)%, or 2 (v / v)%.

[0057] A soil remediation agent according to a third aspect of the present invention comprises the microbial preparation described in the first aspect of the present invention. Since the soil remediation agent employs all the technical solutions of the microbial preparation described in the above embodiments, it possesses at least all the beneficial effects brought about by the technical solutions of the above embodiments.

[0058] The application of the microbial preparation of the first aspect embodiment of the fourth aspect embodiment, the microbial preparation obtained by the preparation method of the first aspect embodiment, or the soil remediation agent of the third aspect embodiment according to any one of A1) to A4): A1) Degradation of carbendazim; A2) Prepare products that degrade carbendazim; A3) Remediation of soil contaminated with carbendazim; A4) Prepare products for remediating soil contaminated with carbendazim; A5) Cultivating edible fungi; A6) Prepare products for cultivating edible fungi.

[0059] According to some embodiments of the present invention, the product includes at least one of reagents and kits.

[0060] According to some embodiments of the present invention, the concentration of carbendazim in the soil is 5 mg / kg to 700 mg / kg. For example, it can be 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, 50 mg / kg, 55 mg / kg, 60 mg / kg, 65 mg / kg, or 70 mg / kg.

[0061] According to some embodiments of the present invention, the edible fungus includes Termitomyces albuminosus.

[0062] A method for degrading carbendazim according to a fifth aspect embodiment of the present invention includes the following steps: The sample containing carbendazim is brought into contact with the microbial preparation described in the first aspect embodiment, the microbial preparation prepared by the preparation method described in the first aspect embodiment, or the soil remediation agent described in the third aspect embodiment.

[0063] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Detailed Implementation

[0064] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0065] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0066] In the description of this invention, the use of terms such as first, second, third, fourth, etc., is merely for the purpose of distinguishing technical features and should not be construed as indicating or implying relative importance, or implicitly indicating the number of technical features indicated, or implicitly indicating the order of the technical features indicated.

[0067] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0068] "And / or" is used to indicate that one or both of the described situations may occur, for example, A and / or B includes (A and B) and (A or B).

[0069] Unless otherwise specified, "about" in this invention means that the allowable error is within ±5%.

[0070] Unless otherwise specified, "room temperature" in this invention means (25±5)℃.

[0071] Unless otherwise specified, the strain information and culture methods in the embodiments and comparative examples of this invention are shown in Table 1.

[0072] Table 1

[0073] Example 1 This example provides a microbial preparation, the preparation method of which is as follows: S1. Preparation of reaction raw materials: (1) Mixed bacterial suspension: Sphingomonas, Burkholderia cepacia, Bacillus amyloliquefaciens, Bacillus megaterium, Trichoderma koningii, Streptomyces, Azotobacter brasiliensis, and Pseudomonas fluorescens, all in the logarithmic growth phase, were centrifuged, washed, and then resuspended in PBS to a bacterial concentration of 1×10⁻⁶. 9 CFU / mL. Sphingomonas, Burkholderia cepacia, Bacillus amyloliquefaciens, Bacillus megaterium, Trichoderma cornutica, Streptomyces, Azotobacter brasiliensis, and Pseudomonas fluorescens were mixed in a ratio of 1:1:1:1:1:1:1:1:1 to obtain a mixed bacterial suspension.

[0074] (2) Sodium alginate aqueous solution: Dissolve an appropriate amount of sodium alginate in deionized water and stir at 60°C to obtain a sodium alginate aqueous solution with a concentration of 3 (w / v)%.

[0075] (3) Carbendazim molecularly imprinted polymer: 0.17 mmol carbendazim, 0.68 mmol methacrylic acid (MAA), and 6 mL acetonitrile were mixed in a 100 mL round-bottom flask and incubated in an ice-water bath for 10 min for prepolymerization; then 3.4 mmol ethylene glycol dimethacrylate (EGDNA) was added, nitrogen was blown for 10 min to remove oxygen, and ultrasonic treatment was carried out under nitrogen for 5 min at a frequency of 50 W; 0.25 mmol azobisisobutyronitrile (AIBN) was added, and the reaction was carried out at 60 °C for 24 h. The supernatant was removed by centrifugation. The precipitate was washed with a methanol / acetic acid mixture (methanol:acetic acid = 9:1 (v:v)) by Soxhlet extraction until carbendazim was undetectable in the supernatant; then washed with methanol to remove residual acetic acid; and dried under vacuum at 45 °C for 24 h to obtain the carbendazim molecularly imprinted polymer.

[0076] (4) Chitosan solution: Mix an appropriate amount of chitosan with a 1 (v / v)% aqueous acetic acid solution and stir to dissolve, so as to obtain a chitosan solution with a final concentration of 0.5 (w / v)%.

[0077] S2. Mix the mixed bacterial suspension with an equal volume of sodium alginate aqueous solution to obtain a mixed solution. At 25°C, use a precision peristaltic pump to drop the mixed solution into a 2 (w / w)% calcium chloride aqueous solution, controlling the flow rate at 2 mL / min. After standing for 30 min to crosslink and solidify, wash with sterile distilled water to remove unreacted substances, obtaining a core with a diameter of approximately 2 mm.

[0078] S3. Mix 20 mg of carbendazim molecularly imprinted polymer with 1 mL of chitosan solution to obtain a coating solution. Immerse 20 mg of the core obtained in step S2 into the coating solution and stir at 25°C for 60 min (stirring speed 20 rpm) to carry out the film-forming reaction. After removing unreacted chitosan by washing with water, dry at room temperature to obtain the microbial preparation.

[0079] Example 2 This example provides a microbial preparation, the preparation method of which is basically the same as that in Example 1, the only difference being the ratio of Sphingomonas, Burkholderia cepacia, Bacillus amyloliquefaciens, Bacillus megaterium, Trichoderma koningii, Streptomyces, Azotobacter brasiliensis, and Pseudomonas fluorescens in the mixed bacterial suspension.

[0080] Specifically, the preparation method of the mixed bacterial suspension is as follows: *Sphingomonas*, *Burkholderia cepacia*, *Bacillus amyloliquefaciens*, *Bacillus megaterium*, *Trichoderma cornutica*, *Streptomyces*, *Azotobacter brasiliensis*, and *Pseudomonas fluorescens*, all in the logarithmic growth phase, are centrifuged, washed, and then resuspended in PBS to a bacterial concentration of 1 × 10⁻⁶. 9CFU / mL. Sphingomonas, Burkholderia cepacia, Bacillus amyloliquefaciens, Bacillus megaterium, Trichoderma cornutica, Streptomyces, Azotobacter brasiliensis, and Pseudomonas fluorescens were mixed in a ratio of 1:3:2:1:0.5:1:3:0.8 to obtain a mixed bacterial suspension.

[0081] Example 3 This example provides a microbial preparation, the preparation method of which is as follows: Sphingomonas, Burkholderia cepacia, Bacillus amyloliquefaciens, Bacillus megaterium, Trichoderma cornigeri, Streptomyces, Azotobacter brasiliensis, and Pseudomonas fluorescens, all in logarithmic growth phase, were centrifuged, washed, and resuspended in PBS to a bacterial concentration of 1 × 10⁻⁶. 9 CFU / mL. The microbial preparation is prepared by mixing *Sphingomonas*, *Burkholderia cepacia*, *Bacillus amyloliquefaciens*, *Bacillus megaterium*, *Trichoderma cornutica*, *Streptomyces*, *Azotobacter brasiliensis*, and *Pseudomonas fluorescens* in a ratio of 1:1:1:1:1:1:1:1:1.

[0082] Comparative Example 1 This example provides a microbial preparation, the preparation method of which is basically the same as that in Example 1, the only difference being that Burkholderia cepacia, Bacillus amyloliquefaciens, Bacillus megaterium, Trichoderma koningii, Streptomyces, Azotobacter brasiliensis, and Pseudomonas fluorescens are omitted.

[0083] Comparative Example 2 This example provides a microbial preparation, the preparation method of which is basically the same as that in Example 1, the only difference being that: Sphingomonas, Bacillus amyloliquefaciens, Bacillus megaterium, Trichoderma koningii, Streptomyces, Azotobacter brasiliensis, and Pseudomonas fluorescens are omitted.

[0084] Comparative Example 3 This example provides a microbial preparation, the preparation method of which is basically the same as that in Example 1, the only difference being that: Sphingomonas, Burkholderia cepacia, Bacillus megaterium, Trichoderma koningii, Streptomyces, Azotobacter brasiliensis, and Pseudomonas fluorescens are omitted.

[0085] Comparative Example 4 This example provides a microbial preparation, the preparation method of which is basically the same as that in Example 1, the only difference being that: Sphingomonas sphingosine monocytogenes, Burkholderia cepacia, Bacillus amyloliquefaciens, Trichoderma koningii, Streptomyces, Azotobacter brasiliensis, and Pseudomonas fluorescens are omitted.

[0086] Comparative Example 5 This example provides a microbial preparation, the preparation method of which is basically the same as that in Example 1, the only difference being that: Sphingomonas, Burkholderia cepacia, Bacillus amyloliquefaciens, Bacillus megaterium, Streptomyces, Azotobacter brasiliensis, and Pseudomonas fluorescens are omitted.

[0087] Comparative Example 6 This example provides a microbial preparation, the preparation method of which is basically the same as that in Example 1, the only difference being: the absence of Sphingomonas sphingosine monocytogenes, Burkholderia cepacia, Bacillus amyloliquefaciens, Bacillus megaterium, Trichoderma koningii, Azotobacter brasiliensis, and Pseudomonas fluorescens.

[0088] Comparative Example 7 This example provides a microbial preparation, the preparation method of which is basically the same as that of Example 1, the only difference being that: Sphingomonas sphingosine monocytogenes, Burkholderia cepacia, Bacillus amyloliquefaciens, Bacillus megaterium, Trichoderma koningii, Streptomyces, and Pseudomonas fluorescens are omitted.

[0089] Comparative Example 8 This example provides a microbial preparation, the preparation method of which is basically the same as that of Example 1, the only difference being: the absence of Sphingomonas sphingosine monocytogenes, Burkholderia cepacia, Bacillus amyloliquefaciens, Bacillus megaterium, Trichoderma koningii, Streptomyces, and Azotobacter brasiliensis.

[0090] Comparative Example 9 This example provides a microbial preparation, the preparation method of which is basically the same as that of Example 1, the only difference being: the absence of Sphingomonas sphingosine monocytogenes, Burkholderia cepacia, Bacillus amyloliquefaciens, Bacillus megaterium, Trichoderma koningii, Streptomyces, Azotobacter brasiliensis, and Pseudomonas fluorescens.

[0091] Comparative Example 10 This example provides a microbial preparation, the preparation method of which is basically the same as that in Example 1, except that Bacillus amyloliquefaciens is replaced with an equal amount of Bacillus licheniformis.

[0092] Comparative Example 11 This example provides a microbial preparation, the preparation method of which is basically the same as that in Example 1, except that Bacillus megaterium is replaced with an equal amount of Bacillus subtilis.

[0093] Comparative Example 12 This example provides a microbial preparation, the preparation method of which is basically the same as that in Example 1, except that Trichoderma corniglita is replaced with an equal amount of Trichoderma harzianum.

[0094] Test Example 1 Soil in which no carbendazim was detected was mixed with an appropriate amount of carbendazim to obtain test soil with a carbendazim concentration of 10 mg / kg. (The last sentence appears to be incomplete and possibly refers to a different topic.) 9 The microbial agents from Examples 1-3, Comparative Examples 1-8, and Comparative Examples 10-13 were added at a CFU / kg test soil concentration. These were then mixed evenly with the test soil to form experimental groups. The microbial agent from Comparative Example 9 was treated similarly and served as the control group. After standing at 25℃ for 15 and 30 days, the concentration of carbendazim in each group's soil was measured, and the relative degradation rate of carbendazim was calculated.

[0095] Relative degradation rate of carbendazim (%) = carbendazim concentration in experimental group / carbendazim concentration in control group × 100%.

[0096] The statistical results of the carbendazim degradation rate of each group after 15 days of treatment are shown in Table 2.

[0097] Table 2

[0098] The relative degradation rates of carbendazim in Example 1, Example 2, and Example 3 after 15 days of treatment were 68.4%, 63.1%, and 61.9%, respectively, and after 30 days of treatment, the relative degradation rates were 90.3%, 84.9%, and 83.7%, respectively. Using any one of the following strains—Sphingomonas, Burkholderia cepacia, Bacillus amyloliquefaciens, Bacillus megaterium, Trichoderma corniganense, Streptomyces, Azotobacter brasiliensis, or Pseudomonas fluorescens—or replacing any one of the following strains with other strains, significantly reduced the relative degradation rate of carbendazim. This indicates that Sphingomonas, Burkholderia cepacia, Bacillus amyloliquefaciens, Bacillus megaterium, Trichoderma corniganense, Streptomyces, Azotobacter brasiliensis, and Pseudomonas fluorescens can synergistically improve the degradation effect on carbendazim.

[0099] Test Example 2 Mix 28% cottonseed hulls, 56% sawdust, 8% wheat bran, 6% rice bran, 1% lime powder, and 1% sucrose, adjust the moisture content to 60%, pack into polyethylene plastic bags (15 cm × 42 cm) at 1.5 kg / bag, sterilize under normal pressure, cool, inoculate with black-skinned chicken mushroom strain, and culture conventionally until the substrate surface is fully covered with mycelium before use.

[0100] Press 10 9 The microbial preparations of Examples 1-2 and Comparative Examples 10-12 were mixed with soil contaminated with Carbendazim (Carbendazim concentration 5 mg / mL) to obtain the casing soil; soil without Carbendazim contamination was used as a blank control group. The sides of the mushroom bags covered with mycelium were cut, and the bags were packed at 16 bags / m² with the cut side facing upwards. 2The fungi were laid flat in sterilized seedbeds, covered with 3 cm of topsoil, and cultured routinely until harvesting. The bioconversion rate was calculated. Soil samples were taken from around the fungi for sequencing and Shannon index analysis to reflect microbial community diversity. A higher Shannon index indicates greater community diversity. The experimental land area for each group was 1 m². 2 Each set up three biological replicates.

[0101] Bioconversion rate (%) = Fresh weight of black-skinned chicken mushroom fruiting bodies (g) / Dry weight of culture medium (g).

[0102] The statistical results are shown in Table 3.

[0103] Table 3

[0104] The microbial agents in Examples 1 and 2 significantly improved the growth of *Termitomyces albuminosus* in soil contaminated with *Trichoderma harzianum*, increased the bioconversion rate, and significantly improved the microbial community diversity of the soil surrounding *Termitomyces albuminosus*. Replacing any of the strains of *Bacillus amyloliquefaciens*, *Bacillus megaterium*, or *Trichoderma koningii* with other strains resulted in a significant decrease in the bioconversion rate and a reduction in the microbial community diversity of the soil surrounding *Termitomyces albuminosus*.

[0105] The embodiments of the present invention have been described in detail above with reference to the examples. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. A microbial preparation, characterized in that, Includes mixed bacteria; said mixed bacteria include Sphingomonas, Burkholderia cepacia, Bacillus amyloliquefaciens, Bacillus megaterium, Trichoderma cornigans, Streptomyces, Azotobacter brasiliensis, and Pseudomonas fluorescens.

2. The microbial preparation according to claim 1, characterized in that, The strain number of *Sphingosine monocytogenes* is CICC 10472; and / or, the strain number of *Burkholderia cepacia* is ATCC 25416; and / or, the strain number of *Bacillus amyloliquefaciens* is DSM 23117; and / or, the strain number of *Bacillus megaterium* is CICC 10044; and / or, the strain number of *Trichoderma corniglansium* is CGMCC 3.11531; and / or, the strain number of *Streptomyces* is ATCC25421; and / or, the strain number of *Azospirillum brasiliensis* is DSM 1690; and / or, the strain number of *Pseudomonas fluorescens* is CICC 23876.

3. The microbial preparation according to claim 1, characterized in that, Based on CFU, the ratio of *Sphingomonas*, *Burkholderia cepacia*, *Bacillus amyloliquefaciens*, *Bacillus megaterium*, *Trichoderma cornutica*, *Streptomyces*, *Azotobacter brasiliensis*, and *Pseudomonas fluorescens* is 1: (0.9-3.1): (0.9-2.1): (0.9-1.1): (0.4-1.1): (0.9-1.1): (0.9-3.1): (0.7-1.1). Preferably, the ratio of Sphingomonas, Burkholderia cepacia, Bacillus amyloliquefaciens, Bacillus megaterium, Trichoderma cornutica, Streptomyces, Azotobacter brasiliensis, and Pseudomonas fluorescens, calculated in CFU, is 1: (0.9-1.1): (0.9-1.1): (0.9-1.1): (0.9-1.1): (0.9-1.1): (0.9-1.1): (0.9-1.1).

4. The microbial preparation according to claim 1, characterized in that, The microbial preparation has a core-shell structure, wherein the core is a calcium alginate ball encapsulating the mixed bacteria; the shell is a chitosan layer; optionally, the chitosan layer encapsulates a carbendazim molecularly imprinted polymer.

5. The method for preparing the microbial preparation according to claim 4, characterized in that, Includes the following steps: A fourth mixture of the bacterial suspension of the mixed bacteria and sodium alginate aqueous solution is added dropwise to a calcium salt aqueous solution, cross-linked and solidified, washed, and a core is obtained; the core is immersed in a coating solution and reacted to obtain the microbial preparation. The coating solution includes carbendazim molecularly imprinted polymer and chitosan.

6. The preparation method according to claim 5, characterized in that, The ratio of the mixed bacteria to sodium alginate is 1×10⁻⁶. 9 CFU: (20 mg-40 mg); And / or, the concentration of sodium alginate in the fourth mixture is 1 (w / v)%-2 (w / v)%.

7. The preparation method according to claim 5, characterized in that, The calcium salt in the calcium salt aqueous solution includes at least one of calcium chloride, calcium nitrate, and calcium sulfate; and / or, the concentration of the calcium salt in the calcium salt aqueous solution is 0.5 (w / w)%-5 (w / w)%.

8. A soil remediation agent, characterized in that, This includes the microbial preparations described in any one of claims 1 to 4 or the microbial preparations prepared by the method described in any one of claims 5 to 7.

9. The use of the microbial preparation according to any one of claims 1 to 4, the microbial preparation obtained by the preparation method according to any one of claims 5 to 7, or the soil remediation agent according to claim 8 in any one of A1) to A4): A1) Degradation of carbendazim; A2) Prepare products that degrade carbendazim; A3) Remediation of soil contaminated with carbendazim; A4) Prepare products for remediating soil contaminated with carbendazim; A5) Cultivating edible fungi; A6) Prepare products for cultivating edible fungi.

10. A method for degrading carbendazim, characterized in that, Includes the following steps: The sample containing carbendazim is brought into contact with the microbial preparation according to any one of claims 1 to 4, the microbial preparation prepared by the preparation method according to any one of claims 5 to 7, or the soil remediation agent according to claim 8.

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