A method and application for reducing pyraclostrobin residues in crops using carboxylated chitosan.

Abstract

This invention discloses a method and application for reducing azoxystrobin residues in crops using carboxylated chitosan. Applying an effective amount of carboxylated chitosan to crops treated with azoxystrobin enhances the activities of superoxide dismutase, cytochrome P450, and glutathione S-transferase in the plant, collectively promoting the degradation of azoxystrobin within the plant. This method is simple to apply, easily integrated with existing agricultural practices, and inexpensive, showing promising prospects for widespread application.

Description

Technical Field

[0001] This invention relates to the field of plant pesticide residue technology, specifically to a method and application of using carboxylated chitosan to reduce pyraclostrobin residues in crops. Background Technology

[0002] The structural formula of azoxystrobin is shown in formula (1). It is a broad-spectrum and highly effective methoxyacrylate fungicide with excellent control efficacy against a variety of plant diseases caused by ascomycetes, basidiomycetes, deuteromycetes, and oomycetes, and is widely used in grain and oil crops. However, its high-frequency and high-dose use has led to increasingly serious residue problems in agricultural products, posing a potential threat not only to non-target organisms but also directly endangering the quality and safety of agricultural products and consumer health.

[0003]

[0004] Currently, methods for removing pesticide residues mainly include physical, chemical, and biological pathways. Physical methods (such as washing and adsorption) and chemical methods (such as ozone and photocatalytic degradation) typically suffer from limited treatment efficiency, high costs, potential impact on agricultural product quality, and the possibility of secondary pollution from the treatment agents themselves; their safety and economic viability need improvement. Biodegradation methods, such as those utilizing specific microorganisms or enzyme preparations, while theoretically more targeted, often face bottlenecks in practical applications, including unstable degradation efficiency, poor adaptability of microorganisms to various environments, and difficulty in colonization and functioning in complex farmland environments.

[0005] Chitosan is a natural cationic alkaline polysaccharide derived from the deacetylation of chitin. It possesses advantages such as good biocompatibility, biodegradability, and low cost, and is currently widely used in water treatment, pharmaceuticals, food, and agriculture. Carboxylated chitosan is a derivative of chitosan obtained through a carboxylation reaction. Its molecular structure contains both amino and carboxyl groups, combining the original biocompatibility and biodegradability of chitosan with the hydrophilicity provided by the carboxymethyl group. CN117296842A discloses the use of chitosan in reducing carbofuran residues in crops. The method involves dissolving carboxylated chitosan into a mother liquor and then spraying it on crop leaves, stems, or applying it to the soil, effectively reducing carbofuran residue levels in crops. CN117296843A discloses the use of chitosan in reducing atrazine damage to plants. This method involves applying chitosan aqueous solutions via root irrigation and spraying, which can alleviate atrazine residues. Both of the aforementioned patents indicate that chitosan can reduce pesticide and herbicide residues, but they do not specify whether chitosan can reduce fungicide residues. CN 119423072A discloses a nano-liquid spray film-forming agent for disease and pest resistance in fruit cultivation and its preparation method, using chitosan as a film-forming agent component. However, this invention primarily provides a protective film on the plant surface to reduce water evaporation and respiration on the fruit surface, preventing damage from pathogens, climatic factors, and mechanical damage; it does not reduce pesticide residues. CN105724377A discloses an antidote for tobacco herbicide damage caused by quinclorac residues and its application method, using chitosan as a soil conditioner to promote crop growth and alleviate herbicide damage. However, to date, no research reports have been found regarding whether chitosan can affect the crop's own metabolic processes of fungicides, and through what mechanism, thereby reducing fungicide residues.

[0006] Therefore, in order to eliminate the problem of pyraclostrobin residues in crops, the inventors of this invention have explored a reference method for reducing fungicide residues in plants by utilizing carboxylated chitosan, a natural substance, under laboratory technical conditions. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the main objective of this invention is to provide a method for reducing azoxystrobin residues in crops using carboxylated chitosan. This method promotes the metabolism of azoxystrobin in crops through exogenous application of carboxylated chitosan, and elucidates the potential mechanism by which chitosan promotes the metabolism of azoxystrobin in crops through enzyme activity tests and 16S rDNA sequencing, providing a theoretical basis and scientific guidance for the control of fungicide residues in plants.

[0008] The first objective of this invention is to provide a method for reducing azoxystrobin residues in crops using carboxylated chitosan, specifically comprising: applying an effective amount of carboxylated chitosan to crops treated with azoxystrobin, thereby promoting the degradation of azoxystrobin in crops by regulating the crop's detoxification enzyme system and microbiome.

[0009] Furthermore, the carboxylated chitosan has a degree of carboxylation greater than or equal to 80% and a viscosity less than or equal to 80 cps.

[0010] Furthermore, the carboxylated chitosan can be applied via foliar application and root application.

[0011] Furthermore, the concentration of the carboxylated chitosan applied is 1-100 mg / L; preferably, the concentration of the carboxylated chitosan applied is 10 mg / L.

[0012] Furthermore, the detoxification enzyme system includes superoxide dismutase, cytochrome P450, and glutathione S-transferase.

[0013] When carboxylated chitosan is applied to crops treated with azoxystrobin, superoxide dismutase and cytochrome P450 in the plants respond rapidly and their activity increases continuously in the early stage (0-3 days), while glutathione S-transferase is activated and enhanced in the later stage (7-14 days). These enzymes work together to promote the degradation of azoxystrobin in plants.

[0014] Furthermore, the microbiome is mainly composed of Pseudomonas species that are significantly enriched after treatment with carboxylated chitosan.

[0015] Furthermore, the crops mentioned are wheat, corn, rapeseed, and peanuts.

[0016] The second objective of this invention is to provide an application of using carboxylated chitosan to reduce azoxystrobin residues in crops, specifically including: applying carboxylated chitosan to crops treated with azoxystrobin, thereby promoting the decomposition of azoxystrobin in crops by regulating the crop's detoxification enzyme system and microbiome.

[0017] Furthermore, the degree of carboxylation of carboxylated chitosan is greater than or equal to 80%, and the viscosity is less than or equal to 80 cps.

[0018] Compared with the prior art, the beneficial effects of the present invention are:

[0019] 1. This invention elucidates for the first time the mechanism by which carboxylated chitosan synergistically promotes pesticide residue degradation through a dual pathway of "regulating the plant detoxification system" and "reshaping the plant microbiome," providing a new scientific perspective and technical approach for the green control of pesticide residues;

[0020] 2. This invention utilizes carboxylated chitosan, a natural product, as an elicitor. It is inherently safe and biodegradable, avoiding the risk of secondary residues that may arise from chemical degradation agents. This method is simple to apply (foliar spraying or root treatment), easily integrated with existing agricultural practices, and is cost-effective, showing promising prospects for application and promotion. Attached Figure Description

[0021] Figure 1 The effects of different concentrations of carboxylated chitosan foliar spraying and root treatment on pyraclostrobin metabolism in wheat.

[0022] Figure 2 The effects of single and combined treatments with 10 mg / L carboxylated chitosan and 5 mg / L azoxystrobin on the activities of superoxide dismutase, cytochrome P450, and glutathione S-transferase in wheat.

[0023] Figure 3 To investigate the effects of single and combined treatments of 10 mg / L carboxylated chitosan and 5 mg / L azoxystrobin on the wheat microbiome and to screen key microorganisms that significantly responded to chitosan treatment.

[0024] Figure 4 The effects of single and combined treatments with 10 mg / L carboxylated chitosan and plant endophyte Pseudomonas sp. EN-10 on the activities of superoxide dismutase, cytochrome P450, and glutathione S-transferase in wheat. Detailed Implementation

[0025] To further understand the present invention, preferred embodiments of the present invention are described below in conjunction with examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, and not for limiting the scope of the claims.

[0026] Key reagents used in this experiment:

[0027] Carboxylated chitosan (carboxylation degree ≥80%; viscosity ≤80cps) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0028] Azoxystrobin standard (98.8%) was purchased from Northern Weiye Measurement Group Co., Ltd.

[0029] Example 1: Effects of carboxylated chitosan on azoxystrobin metabolism in wheat

[0030] Wrap plump wheat seeds, free from mold and damage, in damp gauze and place them in seedling trays filled with shallow water. Incubate in a dark, 30°C incubator for 1 day. Add Hoagland's nutrient solution to the seedling trays up to the gauze level. Place the seedling trays in an artificial climate incubator with the following conditions: temperature 25°C; dark cycle (12h:12h); light intensity 250 μmol / m². 2 / s; maintain relative humidity at 70%, incubate for 6-8 days for later use.

[0031] Foliar treatment: Wheat seedlings with uniform growth were transplanted into a new nutrient solution. Azoxystrobin stock solution dissolved in pure water (5 mg / L; based on field recommended dosage) was sprayed evenly onto the wheat leaves, and allowed to stand for 2 hours to allow for full absorption. A stock solution of carboxylated chitosan was prepared using pure water to a concentration of 10,000 mg / L. The carboxylated chitosan stock solution was diluted with pure water to treatment concentrations of 1, 10, and 100 mg / L. During spraying, foam boards were used to prevent the carboxylated chitosan from entering the nutrient solution. Complete wetting of the leaves was the standard for spraying. The azoxystrobin treatment groups and the control group (without azoxystrobin) were sprayed with the same amount of water. This experiment included four treatment groups, with three replicates for each treatment: a control group (azoxystrobin-1 mg / L chitosan), azoxystrobin-10 mg / L carboxylated chitosan, and azoxystrobin-100 mg / L carboxylated chitosan. Wheat plants were collected at 0, 1, 3, 7, and 14 days of cultivation. The nutrient solution in the roots was washed off, and the residues of azoxystrobin in the plants were measured.

[0032] Root treatment: Azoxystrobin stock solution (5 mg / L; based on field recommended dosage) was evenly sprayed onto wheat leaves and allowed to stand for 2 hours to allow for full absorption by the wheat. A stock solution of carboxylated chitosan (10000 mg / L) was prepared using pure water. This stock solution was then evenly added to Hoagland's nutrient solution to prepare nutrient solutions of 1, 10, and 100 mg / L. Wheat seedlings of uniform growth were transplanted into nutrient solutions containing chitosan (1, 10, and 100 mg / L) and those without chitosan, ensuring full contact between the wheat roots and the nutrient solution. The experimental groups were set up with the same foliar treatment, with three replicates for each treatment. Wheat plants were collected at 0, 1, 3, 7, and 14 days of cultivation. The roots were washed to remove the nutrient solution, and the residual azoxystrobin in the plants was measured.

[0033] like Figure 1 As shown, both foliar spraying and root treatment with carboxylated chitosan significantly promoted the decomposition of azoxystrobin in wheat. The effect of 10 mg / L carboxylated chitosan on the metabolism of azoxystrobin was stronger. After 14 days of foliar spraying and root treatment with chitosan, the concentration of azoxystrobin decreased to 68.1% and 47.7% of the control group, respectively.

[0034] Example 2: Effects of carboxylated chitosan on the enzyme activities of superoxide dismutase (SOD), cytochrome P450 (P450), and glutathione S-transferase (GST) in wheat.

[0035] The experimental treatments were the same as the foliar treatments in Example 1. Four experimental groups were set up, including a blank control group, azoxystrobin treatment group, carboxylated chitosan treatment group, and azoxystrobin + carboxylated chitosan combined treatment group. The concentration of azoxystrobin treatment was 5 mg / L, and the concentration of carboxylated chitosan treatment was 10 mg / L. Each treatment was set up in 3 replicates. Wheat plants were collected on days 3, 7, and 14, and the root nutrient solution was washed off to determine the enzyme activities of superoxide dismutase (SOD), cytochrome P450 (P450), and glutathione S-transferase (GST) in the plants.

[0036] Crude enzyme extraction was performed on plant tissues. The plant tissues were mixed with pre-cooled 0.9% physiological saline at a ratio of 1:9 in centrifuge tubes and homogenized in an ice-water bath at 8000 mL / min. Centrifuge at 10 r / min for 10 min, collect the supernatant, and perform enzyme activity assays according to the enzyme kit instructions. The experimental steps for each enzyme activity assay are the same, but the reagents used are different. The specific steps are as follows: Take out the ELISA plate and let it stand at room temperature for 20 min. Then set up the standard wells and sample wells. Add 50 μL of enzyme standard solution of different concentrations to the standard wells, add 10 μL of the sample to be tested to the sample wells, and then add 40 μL of sample dilution buffer. Then add 100 μL of horseradish peroxidase (HRP) labeled enzyme detection antibody to the standard wells and sample wells. Seal all wells with sealing film and place it in a 37°C water bath for 1 h. Take out the ELISA plate, pour out the liquid, and pat dry on absorbent paper. Then add 300 μL of washing buffer using a multichannel pipette, let stand for 1 min, pour out the liquid, and pat dry on absorbent paper. Repeat 5 times. Then add 50 μL each of substrate A and B for each enzyme to each well and incubate in a 37°C water bath in the dark for 15 min. After min, add 50 μL of stop solution to each well and measure the absorbance of each well at 450 nm using a microplate reader. Substitute the absorbance into the standard curve to calculate the enzyme activity in the sample.

[0037] Superoxide dismutase (SOD) is the first line of defense for plants against fungicide damage. It is primarily responsible for scavenging harmful reactive oxygen species induced by fungicides, reducing cell damage, and activating downstream detoxification enzyme systems to accelerate fungicide metabolism. Cytochrome P450 (P450) and glutathione S-transferase (GST) are the core enzymes for metabolizing fungicides. P450 enzymes, as phase I metabolic enzymes, can chemically modify fungicide molecules through oxidation and other mechanisms, altering their chemical structure and converting them into substances with lower or no toxicity. GST enzymes, as phase II metabolic enzymes, catalyze coupling reactions that allow glutathione to bind to fungicide molecules pre-treated by P450, further detoxifying them. Figure 2As shown, carboxylated chitosan synergistically activates the antioxidant enzyme system and detoxification system of wheat. After treatment with carboxylated chitosan, superoxide dismutase (SOD) and phase I metabolic enzyme P450 respond rapidly and their activity continues to increase in the early stage of azoxystrobin digestion (0-3 d), while phase II metabolic enzyme GST is activated in the later stage of azoxystrobin digestion (7-14 d). These enzymes work together to promote the degradation of azoxystrobin in plants.

[0038] Example 3: Regulatory effect of carboxylated chitosan on the microbiome in wheat

[0039] The carboxylated chitosan leaf treatment and experimental group setup were the same as in Example 2.

[0040] After 7 days of treatment, samples were flash-frozen in liquid nitrogen, ground, and stored at -80°C. Three replicates were performed for each treatment group. Samples were sent to Beijing Novogene Bioinformatics Technology Co., Ltd. for high-throughput sequencing of 16S rRNA genes (V5-V7) using primers 799F / 1193R. Data from each sample were split according to the barcode sequence, and barcode sequences and PCR amplification primers were removed. The raw data were assembled using Flash software, redundant sequences were cut using Cutadapt software, and filtered using Fastp software. Chimera, chloroplast, and mitochondrial sequences were removed by comparison with the Silva database to obtain the final valid data. Noise reduction was performed on the valid data using the DADA2 module in QIIME2 software to obtain the final amplicon sequence variants (ASVs), which were then compared with the Silva database for species annotation. Bacterial community structure changes were analyzed based on species abundance at the phylum and genus levels. The effects of carboxylated chitosan and azoxystrobin on bacterial community β-diversity were investigated using principal coordinate analysis (PCoA). Simper's test was used to screen for ASVs that significantly responded to chitosan treatment.

[0041] like Figure 3 As shown, PCoA analysis revealed that treatment with 10 mg / L carboxylated chitosan significantly remodeled the composition of the wheat microbiome. Based on the Simper test, we identified the ASVs that significantly responded to carboxylated chitosan treatment. Several Pseudomonas strains were significantly enriched after treatment with carboxylated chitosan. We hypothesize that carboxylated chitosan may promote the conversion of azoxystrobin in wheat by enriching beneficial microorganisms such as Pseudomonas.

[0042] Example 4: Carboxylated chitosan drives the enrichment of Pseudomonas sp. EN-10 and synergistically promotes pyraclostrobin metabolism in wheat.

[0043] Isolation of plant endophytic bacteria: The leaf treatment with carboxylated chitosan and the experimental setup were the same as in Example 2. 2.0 g of plant sample was weighed after 7 days of treatment, washed 5 times with sterile water, then washed with 75% alcohol for 1.5 min, disinfected with 2.8% sodium hypochlorite solution for 5 min, and finally washed with sterile water for 1 min. The treated sample was then cut into fragments and ground using a sterile blade. The ground sample was placed in a sterile Erlenmeyer flask, 30 mL of sterile PBS was added, and the mixture was shaken in a constant temperature shaker for 2 h (25℃, 150 r / min). After standing for 30 min, the supernatant was serially diluted (10⁻⁶ ppm). -4 -10 -5 After dilution, 100 μL was evenly spread onto pre-prepared LB, TSA, or R2A agar plates. After complete absorption of the suspension, the plates were incubated for 24 to 72 hours. Single colonies with different morphologies were selected for subculturing, and the purified strains were identified for subsequent experiments.

[0044] Resistant bacterial DNA was extracted and purified using a bacterial DNA extraction kit and used as a template for PCR amplification with universal primers for the 16S rRNA gene. The PCR products were sent to Zhejiang Shangya Biotechnology Co., Ltd. for sequencing. The qualified 16S rRNA gene sequence was compared with the NCBI nucleic acid database using BLASTN to obtain species annotation information.

[0045] The results showed that multiple Pseudomonas endophytic bacteria were isolated from wheat leaves treated with carboxychitosan. Alignment of the 16S rRNA of the isolated endophytic bacteria with the characteristic sequences of carboxychitosan-responsive ASVs identified a plant endophyte, Pseudomonas sp. EN-10, which was significantly enriched after chitosan treatment. Therefore, we selected Pseudomonas sp. EN-10 to verify its role in azoxystrobin metabolism in wheat.

[0046] To investigate the regulatory effect of the plant endophytic fungus *Pseudomonas sp.* EN-10* on plants: Healthy wheat plants with uniform growth were selected and five treatment groups were set up, including a blank control group, azoxystrobin treatment group, azoxystrobin + carboxylated chitosan treatment group, and azoxystrobin + EN-10 treatment group (EN-10 bacterial suspension OD...). 600=0.4 (sprayed evenly) and the azoxystrobin + carboxylated chitosan + EN-10 treatment groups, with azoxystrobin concentration of 5 mg / L and carboxylated chitosan concentration of 10 mg / L, each treatment having 3 replicates. Wheat plants were collected at 0, 1, 3, 7, and 14 days of culture, and the root nutrient solution was washed off to determine the azoxystrobin residue in the plants; wheat plants were also collected at 3, 7, and 14 days of culture, and the root nutrient solution was washed off to determine the activities of superoxide dismutase (SOD), cytochrome P450 (P450), and glutathione S-transferase (GST) in the plants.

[0047] like Figure 4 As shown, the plant endophytic fungus *Pseudomonas sp. EN-10*, similar to carboxylated chitosan, can activate the antioxidant and detoxification enzyme systems in wheat. After 14 days of treatment, the activities of SOD, P450, and GST enzymes in wheat increased by 10.1%, 22.1%, and 11.3%, respectively, compared with the control group. Furthermore, the combined treatment of *Pseudomonas sp. EN-10* and carboxylated chitosan further and efficiently promoted the metabolism of azoxystrobin in wheat. After 14 days of treatment, the activities of SOD, P450, and GST enzymes in wheat increased by 19.2%, 45.6%, and 20.3%, respectively, compared with the control group. Therefore, carboxylated chitosan can regulate the detoxification enzyme system and microbiome of wheat, directly and indirectly promoting the degradation of azoxystrobin in wheat.

Claims

1. A method for reducing azoxystrobin residues in crops using carboxylated chitosan, characterized in that, include: Applying an effective amount of carboxylated chitosan to crops treated with azoxystrobin promotes the degradation of azoxystrobin in crops by regulating the crop's detoxification enzyme system and microbiome.

2. The method according to claim 1, characterized in that, The carboxylated chitosan can be applied via foliar application or root application.

3. The method according to claim 1, characterized in that, The concentration of the carboxylated chitosan applied is 1-100 mg / L.

4. The method according to claim 1, characterized in that, The detoxification enzyme system includes superoxide dismutase, cytochrome P450, and glutathione S-transferase.

5. The method according to claim 1, characterized in that, The crops mentioned are wheat, corn, rapeseed, and peanuts.

6. The method according to claim 1, characterized in that, The carboxylated chitosan has a carboxylation degree of ≥80% and a viscosity of ≤80 cps.

7. An application of carboxylated chitosan to reduce pyraclostrobin residues in crops, characterized in that, Applying carboxylated chitosan to crops treated with azoxystrobin promotes the decomposition of azoxystrobin in crops by regulating the crop's detoxification enzyme system and microbiome; The carboxylated chitosan has a carboxylation degree of ≥80% and a viscosity of ≤80 cps.

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