Application of a strain of the genus xanthomonas in degrading benfluralin and bacterial agent and method

Abstract

The application discloses application of a Flavobacterium strain in degrading benfluralin and a microbial agent and a method. The Flavobacterium strain Flavobacterium glycines sp. nov. can effectively degrade the herbicide benfluralin, can be applied to degrade benfluralin residues in soil, water or farmland environment, repair benfluralin pollution in soil and water, and relieve drug damage of subsequent crops. The application also provides a microbial agent with the strain as an effective component and a method for degrading benfluralin by applying the strain or the microbial agent. The application fills the technical blank of the Flavobacterium strain in degrading benfluralin, has clear degradation effect, is environment-friendly and simple to use, can significantly reduce the risk of benfluralin residues, ensures safety of crops in rotation, and is suitable for repairing herbicide residue pollution and maintaining soil ecological safety in soybean and peanut fields.

Description

Technical Field

[0001] This invention belongs to the field of microbial application technology, and relates to the use of microorganisms to degrade pesticide residues, specifically to the application of a Flavobacterium strain in the degradation of flusulfanilamide, as well as the bacterial agent and method. Background Technology

[0002] Diphenyl ether herbicides are a class of broad-spectrum, highly efficient, and highly selective protoporphyrinogen oxidase (PPO) inhibitors. Due to their strong contact action, low dosage, and broad weed-killing spectrum, they have become a widely used herbicide category in global agricultural production. Currently, commonly used varieties in my country include flufenacet, oxyfluorfen, ethoxyflufenazate, quizalofop-p-ethyl, and trifluralin, mainly applied to soybean, peanut, and other legume fields to control annual broadleaf weeds such as cocklebur, lambsquarters, amaranth, and purslane.

[0003] Because diphenyl ether herbicides are structurally stable, not easily hydrolyzed, and have strong soil adsorption properties, continuous use over many years can easily lead to their accumulation in the soil. Flufenoxuron, one of the most commonly used varieties, has a half-life of 6-12 months in aerobic soils. Even small amounts of residue can cause phytotoxicity to sensitive crops such as corn, rice, sugar beets, watermelons, and vegetables, leading to stunted growth, yellowing, and seedling death, seriously affecting the safety of crop rotation. Simultaneously, herbicide residues can also disrupt the soil microbial community structure, reduce soil enzyme activity, cause soil microecological imbalance, and pose potential environmental risks.

[0004] Microbial degradation is the primary pathway for the decomposition of diphenyl ether herbicides in soil, offering advantages such as environmental friendliness, absence of secondary pollution, and in-situ remediation capabilities, making it an important means of controlling pesticide residue pollution. Currently, various microorganisms capable of degrading diphenyl ether herbicides have been reported both domestically and internationally. These mainly include bacteria such as *Pseudomonas*, *Bacillus*, *Lysinic Bacillus*, *Klebsiella*, *Shigella*, and *Mycobacterium*, as well as fungi such as *Aspergillus* and *White Rot Fungi*. These microorganisms can degrade herbicides such as flufenoxuron and ethoxysulfuron through pathways such as nitro reduction, ether bond cleavage, dechlorination, decarboxylation, and aminoacetylation.

[0005] Although existing research has confirmed the feasibility of microbial degradation, most of the reported degrading strains are common genera such as Pseudomonas and Bacillus, which suffer from problems such as unstable degradation efficiency, weak environmental adaptability, and limited field application effects. More importantly, there are no publicly available reports on the use of Flavobacterium strains for the degradation of flusulfanilamide, nor are there any related products or applications of Flavobacterium strains as microbial agents for the degradation of flusulfanilamide.

[0006] Therefore, developing a Flavobacterium strain with strong environmental adaptability, high degradation efficiency, and good safety, and using it for the degradation and remediation of flusulfanilamide-contaminated soil, and preparing stable and easy-to-use microbial agents, is of great practical significance and application value for solving the problem of herbicide residue damage in soybean fields, ensuring the safety of subsequent crop production, and restoring the farmland soil ecology. Summary of the Invention

[0007] The first objective of this invention is to provide the application of Flavobacterium glycines sp. nov. in the degradation of flumethrin; the second objective is to provide a microbial agent for degrading flumethrin; and the third objective is to provide a method for degrading flumethrin.

[0008] The above-mentioned objective of this invention is achieved through the following technical solution:

[0009] This invention provides the application of the Flavobacterium glycines sp. nov. strain in the degradation of flusulfanilamide. This invention is the first to discover that the known strain Flavobacterium glycines sp. nov. exhibits significant degradation activity against the diphenyl ether herbicide flusulfanilamide, achieving highly efficient degradation of flusulfanilamide and providing a new microbial resource and technical solution for the bioremediation of residual pollution from this type of herbicide.

[0010] Furthermore, the application is to degrade flumetsulam residues in soil, water bodies, or farmland environments. The application scenarios include, but are not limited to: soil in soybean, peanut, and other legume crop fields where flumetsulam has been applied for a long period; polluted water bodies generated during the production or use of flumetsulam; and environmental media surrounding farmland contaminated by this herbicide. This can effectively reduce the residual concentration of flumetsulam in environmental media, thereby reducing its potential risks to the environment and non-target organisms.

[0011] Furthermore, the application is to remediate flusulfanil-contaminated soil and alleviate herbicide damage to subsequent crops. Flusulfanil has a long residual period in the soil and easily causes herbicide damage to sensitive crops such as corn, sugar beets, watermelons, and vegetables. The strain described in this invention can reduce the effective content of flusulfanil in the soil through in-situ degradation, thereby alleviating or eliminating herbicide damage symptoms such as growth inhibition, yellowing, and seedling death in subsequent crops, ensuring the safety of crop production in the crop rotation system, and improving the ecological environment quality of contaminated soil.

[0012] Furthermore, the Flavobacterium glycines sp. nov. is used in the form of live bacteria, fermentation broth, bacterial liquid, bacterial sludge, or freeze-dried powder. All of these forms use Flavobacterium glycines sp. nov., which has degradation activity, as the core component, and can be selected according to the actual application scenario and process requirements. Among them, the live bacteria and fermentation broth forms are suitable for rapid remediation in liquid environments or soil, while the bacterial sludge and freeze-dried powder forms are more conducive to long-term storage, transportation, and industrial preparation of bacterial agents, and all can stably exert the function of degrading flusulfanilamide.

[0013] This invention also provides a microbial agent for degrading flusulfanil, wherein the microbial agent uses Flavobacterium glycines sp. nov. as the active ingredient. This agent, with Flavobacterium glycines sp. nov., which has flusulfanil-degrading activity, as its core functional component, can efficiently degrade flusulfanil through the metabolic activity of the strain, exhibiting both environmental friendliness and safety in use, and can be widely applied to the bioremediation of flusulfanil-contaminated environments.

[0014] Furthermore, the microbial agent also includes an agriculturally acceptable carrier and is formulated into an agriculturally acceptable formulation. The carrier provides a protective, dispersing, and slow-release environment for the microbial strain, improving its survival rate and stability in the environment and reducing the impact of adverse environmental factors on its activity. By combining the carrier with the formulation, the storage stability, transportation convenience, and field application effect of the microbial agent can be significantly improved, meeting the application needs in different scenarios.

[0015] Furthermore, the carrier is selected from one or more of diatomaceous earth, talc, calcium carbonate, humic acid, zeolite powder, wheat bran, and starch. These carriers are all commonly used inert or functional carriers in the agricultural field, characterized by low cost, good environmental compatibility, and no secondary pollution. Among them, inorganic carriers such as diatomaceous earth, talc, and calcium carbonate can be used as inert fillers to disperse bacterial strains, while organic carriers such as humic acid, wheat bran, and starch can also serve as auxiliary carbon sources, providing nutritional support for the colonization and growth of bacterial strains in the soil, further enhancing the application effect of the microbial agent.

[0016] Furthermore, the microbial agent formulation is in the form of powder, wettable powder, aqueous solution, suspension concentrate, granules, or water-dispersible granules. Different formulations are suitable for different application scenarios and equipment: powders and wettable powders are easy to spread or mix with soil; aqueous solutions and suspension concentrates are suitable for fertigation, drip irrigation, or spraying; granules can be applied evenly over a large area through mechanized spreading; and water-dispersible granules combine dispersibility and storage stability, allowing for flexible selection based on different crop field cultivation methods and application habits.

[0017] This invention also provides a method for degrading flumethrin residues in soil, water, or farmland environments by applying an effective amount of Flavobacterium glycines sp. nov. or any of the aforementioned microbial agents to the contaminated system. The method introduces a Flavobacterium glycines sp. nov. strain or its agent with degradation activity into the flumethrin-contaminated soil, water, or farmland environment, allowing the strain to colonize the contaminated system and exert metabolic degradation effects, thereby reducing the residual concentration of flumethrin and achieving pollution remediation. This method is simple to operate, requires no complex equipment, can be implemented in situ, does not damage the original soil or water structure, and poses no risk of secondary pollution.

[0018] Furthermore, the application methods include broadcasting, furrow application, hole application, fertigation, drip irrigation, or soil mixing. These application methods are all conventional field operations in agricultural production and can be flexibly selected based on the type of pollutant, crop planting pattern, and inoculant formulation: broadcasting, furrow application, hole application, and soil mixing are suitable for soil environment treatment, while fertigation and drip irrigation are more suitable for paddy fields, facility agriculture, or large-scale farmland, ensuring that the bacterial strain or inoculant is evenly distributed in the polluted system and fully exerts its degradation effect.

[0019] Beneficial effects:

[0020] 1. This invention discloses for the first time that the Flavobacterium glycines sp. nov. has degradation activity against flusulfanil, filling the gap in the prior art where there is no Flavobacterium strain that can degrade flusulfanil, and providing a new microbial resource for the management of diphenyl ether herbicide residues.

[0021] 2. The experiments of this invention show that the strain Flavobacterium glycines sp. nov. can achieve a degradation rate of 36.78% for 50 mg / L flumethrin under neutral conditions after 27 h of culture. The degradation effect is stable and the environmental adaptability is strong, which can effectively reduce the residual amount of flumethrin in soil and water.

[0022] 3. The microbial inoculant provided by this invention uses Flavobacterium glycines sp. nov. as the active ingredient, is compatible with agriculturally acceptable carriers, has diverse formulations, is easy to apply, and is suitable for large-scale field application. This invention achieves in-situ degradation by applying the strain or inoculant, is simple to operate, has no secondary pollution, and can significantly alleviate the phytotoxicity of flusulfanil residues to subsequent sensitive crops, ensuring the safety of crop rotation production. Attached Figure Description

[0023] Figure 1This is a colony morphology diagram of Flavobacterium glycines sp. nov. Detailed Implementation

[0024] The substantive content of the present invention will be described in detail below with reference to specific embodiments. However, those skilled in the art should know that the scope of protection of the present invention should not be limited to these specific embodiments.

[0025] Example 1: Degradation activity of Flavobacterium glycines sp. nov. against flusulfanilamide

[0026] I. Experimental Materials

[0027] Flavobacterium glycines sp. nov. was purchased from Wuhan Gray Algae Biotechnology Co., Ltd. (accession number NBRC 105008, also deposited in ICMP 17618).

[0028] Inorganic salt liquid culture medium: 6.8g Na₂HPO₄, 3.0g KH₂PO₄, 0.5g NaCl, 1.0g (NH₄)₂SO₄, 0.5g MgSO₄·7H₂O, 0.02g CaCl₂·2H₂O, 10g glucose, and distilled water to a final volume of 1000 mL. Mix thoroughly and adjust the pH to 7.0. Autoclave (121 ℃, 20 min) for later use. Simultaneously add autoclaved trace elements: 0.1 mg / L ZnSO₄·7H₂O, 0.03 mg / L MnCl₂·4H₂O, 0.01 mg / L CuSO₄·5H₂O, 0.02 mg / L Na₂MoO₄·2H₂O, 0.005 mg / L CoCl₂·6H₂O, and 1.0 mg / L FeSO₄·7H₂O. Mix and set aside.

[0029] II. Experimental Methods and Results

[0030] Flavobacterium glycines sp. nov. strain (e.g.) on the plate Figure 1 The culture was inoculated into an inorganic salt liquid medium and cultured in the dark with shaking at 30°C and 160 rpm until the logarithmic growth phase to obtain a bacterial suspension. The OD of the bacterial suspension was adjusted with sterile water. 600=1, 10% of the inoculum was inoculated into an inorganic salt liquid medium with a flumetsulam concentration of 50 mg / L, and cultured in the dark with shaking at 30℃ and 160 r / min. Samples were taken 27 h after inoculation, and the residual concentration of flumetsulam in the culture medium was determined by high performance liquid chromatography (HPLC). A sterile inorganic salt liquid medium containing flumetsulam at the same mass concentration was used as a blank control, and each treatment was repeated in triplicate.

[0031] The detection method for flusulfanilamide is as follows: Take 2 mL of flusulfanilamide-containing culture medium from different treatment liquids, dilute it to 20 mL with sterile water, and transfer it to a 250 mL separatory funnel. Extract it three times with 10 mL of dichloromethane. After dehydration of the lower organic phase with anhydrous sodium sulfate, combine the samples into a 50 mL flat-bottom sample bottle, dry it to near dryness by nitrogen blowing on a rotary evaporator, add 5 mL of chromatographic methanol to redissolve it, and then bring the volume to 1 mL by nitrogen blowing again for high performance liquid chromatography analysis.

[0032] The degradation rate of flusulfanilamide is calculated using the following formula:

[0033] Degradation rate (%) = [1 - (Measured residue in treatment group / Measured residue in control group)] × 100%

[0034] The results showed that Flavobacterium glycines sp. nov. had a significant degradation effect on flusulfanilamide, with a degradation rate of 36.78% ± 7.70% after 27 hours.

[0035] Example 2: Microbial inoculant for degrading flusulfanil

[0036] This embodiment provides a microbial agent for degrading flusulfanil residues in soil, water, or farmland environments. The microbial agent uses Flavobacterium glycines sp. nov. as the active ingredient and also contains an agriculturally acceptable carrier (selected from one or more of diatomaceous earth, talc, calcium carbonate, humic acid, zeolite powder, wheat bran, and starch) to form an agriculturally acceptable formulation (powder, wettable powder, aqueous solution, suspension concentrate, granules, or water-dispersible granules).

[0037] The purpose of the above embodiments is to specifically illustrate the substantive content of the present invention, but those skilled in the art should know that the scope of protection of the present invention should not be limited to the specific embodiments.

Claims

1. Application of Flavobacterium glycines sp. nov. in the degradation of flusulfanilamide.

2. The application according to claim 1, characterized in that: The application is to degrade flusulfanilamide residues in soil, water, or farmland environments.

3. The application according to claim 1, characterized in that: The application is to remediate flusulfanil-contaminated soil and alleviate herbicide damage to subsequent crops.

4. The application according to claim 1, characterized in that: Flavobacterium glycines sp. nov. is used in the form of live bacteria, fermentation broth, bacterial liquid, bacterial sludge, or freeze-dried powder.

5. A microbial inoculant for degrading flusulfanilamide, characterized in that: The microbial agent uses Flavobacterium glycines sp. nov. as its active ingredient.

6. The microbial agent according to claim 5, characterized in that: The microbial agent also includes an agriculturally acceptable carrier, forming an agriculturally acceptable microbial agent formulation.

7. The microbial agent according to claim 6, characterized in that: The carrier is selected from one or more of diatomaceous earth, talc, calcium carbonate, humic acid, zeolite powder, wheat bran, and starch.

8. The microbial agent according to claim 6, characterized in that: The microbial agent formulation is a powder, wettable powder, aqueous solution, suspension, granule, or water-dispersible granule.

9. A method for degrading flusulfanilamide residues in soil, water, or farmland environments, characterized in that: Apply an effective amount of Flavobacterium glycines sp. nov. or any of the microbial agents according to claims 5-8 to the contaminated system.

10. The method according to claim 9, characterized in that: The application methods include spreading, trenching, hole application, fertigation, drip irrigation, or mixing with soil.

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