Application of a strain of Trichoderma monochromatum Y4941 in the degradation of imidacloprid pesticides

Abstract

This invention belongs to the field of environmental microbiology and utilizes microbial methods to degrade chemical herbicide residues. Specifically, it relates to the application of a *Cerrena unicolor* strain in the degradation of imidacloprid pesticide residues. The *Cerrena unicolor* strain is strain Y4941; this strain was deposited on January 24, 2014, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC NO. 8784. The degrading microbial agent of this invention can efficiently degrade imidacloprid pesticide residues in water bodies and on the surface of agricultural products, protecting and restoring the ecological environment.

Description

Technical Field

[0001] This invention belongs to the field of environmental microbiology and utilizes microbial methods to degrade chemical herbicide residues. It involves the application of a strain of Cerrena unicolor in the degradation of imidacloprid pesticide residues. Background Technology

[0002] In the pesticide market, herbicides account for the largest market share. However, herbicides are characterized by high toxicity, long residual time, and difficulty in degradation. Large-scale use of herbicides not only pollutes land and crops but also causes serious pollution to terrestrial water bodies and marine environments. Therefore, exploring effective methods for degrading herbicide residues has become an important long-term research focus. In this field, the biodegradation of pesticide residues using microorganisms has become a hot research area. Microorganisms can break down recalcitrant pesticides into small molecules or oligomers, which are then transformed into easily absorbed and utilized active components through a certain reaction process. This technology can decompose large molecules in organic pollutants into low-molecular-weight compounds. After these compounds are decomposed and absorbed by microorganisms, they are transformed into environmentally harmless, bio-inert substances. Microorganisms can also decompose some water-insoluble organic matter, such as pesticide residues. Due to their small size, rapid reproduction capacity, and large specific surface area, microorganisms are better adapted to their living environment than other organisms. Microorganisms can generate new strains through natural mutation, form new enzyme systems, and discover new metabolic pathways, thereby participating in the degradation and transformation of pesticides and other organic pollutants. Therefore, microorganisms have enormous potential in degrading pesticide residues.

[0003] Research on microbial degradation of pesticides mainly focuses on the screening, isolation, identification, and degradation characteristics of strains, and some progress has been made. Bacteria play an important role in current bioremediation research, but their practical application is limited by adverse effects such as potential groundwater pollution, plant disease, and excessive proliferation clogging soil capillaries and hindering water absorption. Compared to bacteria, fungi have advantages such as the ability to coexist with other microorganisms and maintain a competitive advantage; non-specificity; a wide range of potential substrates; low cost; no secondary pollution; and good ecological restoration. However, fungi are diverse, and their degradation capabilities for environmental pollutants vary greatly. Therefore, obtaining highly efficient pesticide-degrading strains and applying them in practice is of practical significance for reducing the environmental harm caused by pesticide residues. Summary of the Invention

[0004] The purpose of this invention is to provide the application of *Tetranychus monochromatus* strains in the degradation of imidacloprid residues.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] Application of a *Cerrena unicolor* strain in the degradation of pesticide residues in imidacloprid, wherein the *Cerrena unicolor* strain is *Cerrena unicolor* strain Y4941; this strain was deposited at the China General Microbiological Culture Collection Center on January 24, 2014, with accession number CGMCC NO.8784, address of the depository: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0007] The strain was applied to the sample to be treated for co-culture, which degraded and removed imidacloprid from the sample.

[0008] The strain is

[0009] (1) Activation of strain: Select the above-mentioned Trichoderma monochromatum strain and inoculate it into a 70 mm petri dish containing isolation and preservation medium, and culture it at 28-30℃ for 5-7 days; break the cultured strain into mycelial fragments with a diameter of 1 cm;

[0010] (2) Put 100 mL of liquid culture medium into a 250 mL Erlenmeyer flask, autoclave at 121 °C for 30 minutes, cool, inoculate with mycelial fragments under sterile conditions, and culture at 28-30 °C and 120-140 rpm for 5-7 days.

[0011] (3) Homogenize the fermentation broth prepared in the above steps together with the mycelial balls using an internal homogenizer at 3000 rpm / min for 15-20 seconds to obtain the liquid inoculum.

[0012] (4) Cut the straw into sections about 0.5-1cm long, soak them in distilled water for 12 hours, then drain them until they no longer drip water. Pack them into 13*9cm nylon mesh bags (dry weight 15-20g), autoclave at 121℃ for 30 minutes, and after cooling, add liquid bacterial agent under sterile conditions. The inoculation amount is 20-25%.

[0013] (5) Place the nylon mesh bag in a sterile, sealed plastic box and incubate at 28-30℃ for 30 days to obtain solid fermentation agent.

[0014] The liquid or solid fermentation agent in the sample to be treated is added at a rate of 90-110 mL; the solid fermentation agent is added at a rate of 80-100 g / L.

[0015] Furthermore, in a sample containing imazapyr pesticide at a final concentration of 10-20 mg / L, add 100 mL of liquid microbial agent or 100 g / L of rice straw solid fermentation microbial agent, and incubate at 28-30℃ and 120 rpm / min on a shaker for 36 hours.

[0016] The isolation and preservation culture medium consisted of 20g of malt extract powder, 15g of agar, water added to 1L, pH set to natural, and autoclaved at 121℃ for 30 minutes.

[0017] The liquid culture medium consisted of 5g yeast extract, 2g KH₂PO₄, 0.5g MgSO₄, 0.1g CaCl₂, 0.1g CuSO₄·5H₂O, 0.01g vitamin B1, 0.5g Tween 80, with a natural pH. Water was added to bring the volume to 1L, and the mixture was autoclaved at 121°C for 30 minutes.

[0018] The solid fermentation medium for rice straw consists of 1000g of rice straw segments, each segment about 0.5-1cm in length, soaked in distilled water for 12 hours, drained until no water drips, with a natural pH value, and then autoclaved at 121℃ for 30 minutes.

[0019] The degradation rate (%) = (AB) / A × 100,

[0020] Where A represents the pesticide residue value of imidacloprid in the control group.

[0021] B represents the pesticide residue value of imazapyr after degradation by the degrading agent.

[0022] Advantages of this invention: The *Trichoderma monochromatum* strain of this invention is a strain with high vitality, simple cultivation method, fast growth rate, and low mutation rate. Experiments show that this strain can grow in water agar medium using imidacloprid as the sole carbon and energy source and degrade imidacloprid. In a liquid containing 10 mg / L imidacloprid pesticide, the addition of liquid bacterial agent resulted in a degradation rate of over 85% of imidacloprid after 36 hours of co-cultivation. This fully demonstrates that this strain can be used for the biodegradation of imidacloprid residues in water bodies and on the surface of agricultural products, showing promising prospects for industrial application in pesticide residue biodegradation. It can also serve as a model strain for studying the degradation mechanism of imidacloprid pesticide residues by white-rot fungi. Attached Figure Description

[0023] Figure 1 The liquid chromatogram of a 10 mg / L concentration of imidacloprid provided in Example 2 of the present invention;

[0024] Figure 2 This is the liquid chromatogram of the blank control group, *Imidacloprid*, provided in Example 2 of the present invention;

[0025] Figure 3 This is a liquid chromatogram of imidacloprid smoke after degradation by the degrading agent provided in Example 2 of the present invention; Detailed Implementation

[0026] The bacterial strain that degrades imidacloprid pesticide residues provided in this invention was isolated from decaying wood in virgin forests. The isolation method for this strain is as follows:

[0027] The following detailed examples illustrate the facts of the present invention in order to help readers better understand the spirit and essence of the invention, but are not intended to limit the scope of the invention.

[0028] 1. Culture medium preparation:

[0029] (1) Culture medium for preserving microorganisms (solid, 1L): 20g malt extract, 20g glucose, 15g agar, added to 1L, pH value natural;

[0030] (2) Culture medium for activating microorganisms (solid, 1L): 20g malt extract powder, 15g agar, add water to 1L, pH value is natural;

[0031] (3) Liquid culture medium (liquid, 1L): 5g yeast extract, 2g KH2PO4, 0.5g MgSO4, 0.1g CaCl2, 0.1g CuSO4·5H2O, 0.01g vitamin B1, 0.5g Tween 80, pH value natural, add water to 1L;

[0032] (4) Rice straw solid fermentation culture medium: 1000g of rice straw segments, about 0.5-1cm in length, soaked in distilled water for 12 hours, then drained until no water drips, with a natural pH value.

[0033] All the above culture media were sterilized in an autoclave at 121°C for 30 minutes.

[0034] 2. Activation of the strain: Select hyphae of Trichoderma monochromatum and inoculate them into a 70 mm Petri dish containing strain preservation medium. Incubate at 28°C for 7 days.

[0035] Example 1: Culture of bacterial cells:

[0036] (1) Liquid bacterial agent: Using an inoculation needle, pick up the hyphae of *Cerrena unicolor* and inoculate them into a Petri dish containing the above-mentioned activated culture medium. Incubate at 28℃ for 7 days. The colonies are loose, snow-white, and cotton-like. Inoculate a 1cm piece of the fungus into an Erlenmeyer flask containing 100mL of culture medium that has been autoclaved at 121℃ and 0.1MPa for 30min. Then place the flask in a shaker at 28℃ and 120rpm for 5 days. Homogenize the mixture for 20s using an internal homogenizer to obtain a culture medium containing the extracellular degrading enzymes secreted by the fungus. This is the liquid bacterial agent.

[0037] (2) Solid fermentation inoculant: Add the above liquid inoculant to the rice straw solid fermentation medium at an inoculation rate of 20%, and incubate for 30 days at 28°C in a sterile plastic box to obtain the rice straw solid fermentation inoculant.

[0038] Example 2: Degradation of imidacloprid pesticide residues in liquid by liquid microbial agents

[0039] (1) In Example 1, 100 mL of liquid fungal agent of Trichoderma monocolor was added to the culture medium to be treated with a final concentration of 10 mg / L imidacloprid pesticide, and the medium was shaken at 28°C and 120 rpm. The imidacloprid solution without the addition of liquid fungal agent was used as the control group.

[0040] (2) Samples were taken every 12 hours. 50 mL of sample was placed in a 250 mL separatory funnel, 50 mL of dichloromethane was added, and the mixture was shaken for 15 minutes. The mixture was allowed to stand until the layers separated, and the lower organic phase was collected. This process was repeated three times. The lower organic phase solutions were combined, and the pesticide residues in the culture medium were extracted. The solution was concentrated using a rotary evaporator at 40°C, dissolved in a small amount of dichloromethane, purified using an SPE-anhydrous sodium sulfate column, and analyzed by high-performance liquid chromatography (HPLC). The degradation rate was calculated as follows: Degradation rate (%) = (AB) / A × 100, where A is the pesticide residue value of the control group (9.47 mg / L after 36 hours), and B is the pesticide residue value of the control group after degradation by the liquid bacterial agent (1.36 mg / L after 36 hours). Under these culture conditions, the degradation rate of high-concentration imidacloprid residues by *Cerrena unicolor* Y4941 reached over 85% within 36 hours (see [reference]). Figure 1-3 ).

[0041] Example 3: Degradation of imidacloprid pesticide residues in liquid by solid-state fermentation inoculants

[0042] (1) Add 100g / L of solid fermentation degradation agent of rice straw to 10mg / L of imidacloprid pesticide, and incubate in a 28-30℃ incubator. Use imidacloprid solution without rice straw solid fermentation degradation agent as control group.

[0043] (3) Take 15 mL of sample and place it in a 50 mL plastic centrifuge tube. Add 15 mL of dichloromethane, shake for 15 min, and let stand until the layering is complete. Centrifuge and collect the lower organic phase. Repeat three times, combine the lower organic phase solutions, extract the pesticide residue of imidacloprid, concentrate it in a rotary evaporator at 40 °C, dissolve it in a small amount of dichloromethane, purify it through an SPE-anhydrous sodium sulfate column, and perform high performance liquid chromatography analysis to calculate the degradation rate. The degradation rate (%) = (AB) / A × 100, where A is the pesticide residue value of imidacloprid in the control group, and B is the pesticide residue of imidacloprid after degradation by the degrading agent.

Claims

1. The application of a strain of *Trichoderma monochromatum* in the degradation of pesticide residues in imidacloprid, characterized by: The strain of *Trichoderma monochromatum* is *Trichoderma monochromatum* ( Cerrena unicolor Strain Y4941; This strain was deposited at the China General Microbiological Culture Collection Center on January 24, 2014, with accession number CGMCC NO.8784.

2. The application according to claim 1, characterized in that: The strain was applied to the sample to be treated for co-culture, which degraded and removed imidacloprid from the sample.

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