Use of chitosan in reducing the phytotoxicity of atrazine

Abstract

The application discloses an application of chitosan in reducing the atrazine phytotoxicity of plants. In the research, it is found that a chitosan water agent has the effect of relieving the atrazine phytotoxicity of plants. The application mode of the chitosan water agent includes root irrigation and spraying, and the phytotoxicity relieving effect is increased with the increase of the concentration. Transcriptome sequencing is carried out to explore the influence mechanism. In the soil culture condition, multiple spraying can well relieve the atrazine phytotoxicity of rapeseed. Therefore, the chitosan water agent has important significance for relieving the atrazine phytotoxicity of plants.

Description

Technical Field

[0001] This invention relates to a method for reducing pesticide damage, specifically to a novel use of chitosan in reducing pesticide damage to plants and a method for reducing pesticide damage to plants. Background Technology

[0002] Chemical control plays a crucial role in controlling crop diseases, and pesticides have a positive effect on the healthy growth of crops. Survey data shows that crops using pesticides experience at least a 7% increase in yield annually. Therefore, the use of pesticides is unavoidable in the development of modern agriculture. However, with the widespread use of pesticides, their toxicity and the resulting environmental pollution have gradually attracted attention. Currently, the situation of pesticide use in my country can be summarized as: many types, large amounts, and high residues. According to the Ministry of Agriculture and Rural Affairs of China, the pesticide utilization rate in my country in 2020 was only 40.6%, which is not high. In the actual application process, problems such as not grasping the timing of application, excessive application frequency, and failure to prepare pesticides according to instructions often lead to large amounts of pesticide residues remaining in the current crop and even the soil, thus affecting the growth of subsequent crops.

[0003] With the advancement of domestic herbicide research and development technology and the mastery of field application techniques, in the early years of this century, due to the further expansion of the planting area of ​​oil crops such as soybeans and peanuts, the weed composition in the fields changed, with the occurrence of broadleaf weeds and grass weeds becoming increasingly serious.

[0004] Atrazine [chemical name 2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine] is a triazine herbicide. It is one of the most widely used pesticides globally, used to control broadleaf and grassy weeds in fields such as corn, cotton, rapeseed, sugarcane, and pineapple. Atrazine is a selective endophytic pre-emergence herbicide that controls weed growth by blocking plastoquinone-binding proteins in chloroplasts and inhibiting photosynthesis. Due to its high efficiency, low toxicity, and low cost, it is widely used worldwide and is one of the most commonly used herbicides. In recent years, the increase in noxious weeds has led to a significant increase in atrazine use, resulting in large amounts of atrazine residues in the soil. This not only pollutes the soil environment but also causes damage to many sensitive subsequent crops, leading to reduced yields or even crop failure, seriously affecting the adjustment of agricultural planting structures and the safety of agricultural production. Therefore, research on reducing the phytotoxicity of atrazine to subsequent crops is of great significance for agricultural production and environmental protection. Summary of the Invention

[0005] Chitosan is the only naturally occurring cationic basic polysaccharide, obtained by hydrolyzing chitin (also known as chitin) to remove acetylation groups. Its physicochemical properties are affected by the degree of deacetylation. Chitosan (including its modified derivatives) is widely used in food, medicine, agriculture, and environmental fields due to its multifunctionality, good biocompatibility, and low cost and availability. As a biodegradable and non-toxic green material, it is commonly used in agriculture as a soil conditioner, fruit and vegetable preservative, biodegradable mulch film, and pesticide loading material. However, there are no reports on chitosan reducing pesticide damage.

[0006] Rapeseed, as an important economic crop, suffers greatly from herbicide damage. Therefore, a highly efficient and low-cost method for applying chitosan has been developed to solve the problem of atrazine damage in rapeseed and to provide more effective ways to reduce herbicide damage to crops.

[0007] This study found that chitosan aqueous solution can alleviate atrazine damage in plants. The application methods include root irrigation and spraying, and the mitigation effect increases with increasing concentration. Transcriptome sequencing was performed to explore its mechanism of action. Furthermore, multiple sprayings under soil cultivation conditions effectively alleviated atrazine damage in rapeseed. Therefore, chitosan aqueous solution is of significant importance in alleviating atrazine damage in plants.

[0008] Based on this, one objective of this application is to provide a new use of chitosan in reducing atrazine damage to plants; another objective of this application is to provide a new method for reducing atrazine damage to plants.

[0009] The use of chitosan in reducing atrazine damage to plants.

[0010] Optionally, the structural formula of the chitosan is:

[0011]

[0012] Optionally, the degree of deacetylation of the chitosan is 55%-100%.

[0013] Most preferably, the chitosan has a molar mass of 1526.5 g / mol and a molecular formula of C2. 56 H 103 N9O 39 .

[0014] Most preferably, the degree of deacetylation of the chitosan is greater than 80%.

[0015] Optionally, chitosan is formulated into a chitosan aqueous solution and the chitosan aqueous solution is applied to plants grown in atrazine-contaminated soil or nutrient solution.

[0016] Optionally, the plant may be rapeseed, rice, tomato, or cabbage grown in atrazine-contaminated soil or nutrient solution. More preferably, the plant may be rapeseed grown in atrazine-contaminated soil or nutrient solution.

[0017] Optionally, the application method is irrigation or spraying; the concentration of the chitosan aqueous solution is 1-100 mg / kg; the chitosan aqueous solution is applied 1-4 times, with the first application time being 4-6 days after the seedlings are transplanted into the atrazine-contaminated soil, and the interval between each application being 4-6 days.

[0018] The irrigation method should be such that the soil around the plant roots is moistened, and the spraying should be such that the entire leaf surface is covered.

[0019] More preferably, the concentration of the chitosan aqueous agent is 5–20 mg / kg.

[0020] Most preferably, the concentration of the chitosan aqueous solution is 10 mg / kg, and the chitosan aqueous solution is applied 3 times. The first application is on the 5th day after the seedlings are transplanted into the atrazine-contaminated soil, and the interval between each application is 5 days.

[0021] This application also provides a method for reducing atrazine damage to plants, including:

[0022] Chitosan was formulated into a chitosan aqueous solution, which was then applied to plants grown in atrazine-contaminated soil.

[0023] The plant can be a soil-grown or hydroponic plant. For hydroponic plants, the chitosan aqueous solution can be added directly to the nutrient solution or sprayed onto the surface of the plant leaves. For soil-grown plants, the chitosan aqueous solution can be directly watered into the cultivation soil or sprayed onto the surface of the plant leaves.

[0024] Taking rapeseed as an example, the specific steps include: treating rapeseed with the chitosan agent in different ways and culturing it for a period of time.

[0025] In this invention, chitosan aqueous solution can rapidly and efficiently reduce the atrazine content in rapeseed through root irrigation, alleviating the harmful effects of atrazine on rapeseed. The above aqueous solution was applied to hydroponic rapeseed plants by root irrigation at doses of 1, 10, and 100 mg / kg. After 10 days of cultivation, the atrazine content in rapeseed treated with 1, 10, and 100 mg / kg chitosan was 84.17%, 69.07%, and 61.92% of that in rapeseed without chitosan, respectively. In terms of fresh weight, the rapeseed treated with 1, 10, and 100 mg / kg chitosan was 75.96%, 78.99%, and 79.41% of that in the control group, respectively, while the fresh weight of the atrazine-only treatment group was 56.80% of that in the control group.

[0026] Compared with the prior art, this application has at least one of the following beneficial effects:

[0027] (1) This application found that spraying soil-grown rapeseed with chitosan aqueous solution can significantly restore various biological indicators (plant height and fresh weight) of rapeseed, and is harmless to rapeseed.

[0028] (2) The chitosan aqueous solution of this application can quickly and efficiently reduce the residue of atrazine in rapeseed and alleviate the phytotoxicity of rapeseed. The preparation process of this aqueous solution is simple, the production cost is low, and the use method is convenient, and it has broad application prospects.

[0029] (3) The chitosan aqueous solution in this application can quickly and efficiently reduce the content of atrazine in rapeseed by spraying, thereby alleviating the harmful effects of atrazine on rapeseed. The above aqueous solution was sprayed onto hydroponic rapeseed plants at concentrations of 1, 10, and 100 mg / kg. After 10 days of cultivation, the atrazine content of rapeseed treated with 1, 10, and 100 mg / kg chitosan spraying was 85.37%, 84.12%, and 83.99% of that without chitosan, respectively. In terms of fresh weight, the rapeseed treated with 1, 10, and 100 mg / kg chitosan root irrigation was 74.38%, 72.53%, and 75.15% of that of the control group, respectively, while the fresh weight of the atrazine-only treatment group was 46.76% of that of the control group. Attached Figure Description

[0030] Figure 1 The graph shows the effect of chitosan aqueous solution on the physiological indicators of hydroponic rapeseed plants and the atrazine residue under the condition of root irrigation (A: fresh weight, B: plant height, C: residue).

[0031] Figure 2 The effect of chitosan aqueous solution on the physiological indicators of hydroponic rapeseed plants and the atrazine residue under spray application conditions is shown in the figure (A: fresh weight, B: plant height, C: residue).

[0032] Figure 3 The effect of atrazine residue on rapeseed after three days of exposure to atrazine followed by transplanting into a culture medium containing chitosan aqueous solution;

[0033] Figure 4 The effect of chitosan aqueous solution on important metabolic enzymes of hydroponic rapeseed under root irrigation application conditions is shown in the figure (A: cytochrome P450 enzyme, B: glutathione-S-transferase, C: glycosyltransferase, D: superoxide dismutase, E: peroxidase, F: glutathione reductase).

[0034] Figure 5 The effect of chitosan aqueous solution on important metabolic enzymes of hydroponic rapeseed under spray application conditions (A: cytochrome P450 enzyme, B: glutathione-S-transferase, C: glycosyltransferase, D: superoxide dismutase, E: peroxidase, F: glutathione reductase).

[0035] Figure 6 Volcano plots of differential genes after root irrigation with chitosan aqueous solution: CTS vs CK (A), ATR vs CK (B), and ATR+CTS vs CK (C). Blue represents downregulated genes, red represents upregulated genes, and gray represents genes that meet the criteria with no significant differences.

[0036] Figure 7 Venn diagram comparing the three treatment groups with the control group after root irrigation with chitosan aqueous solution;

[0037] Figure 8 Figure (B) shows the common or unique atrazine detoxification enzymes (DEGs) and shared detoxification enzymes (DEGs) among different treatment groups after root irrigation with chitosan aqueous solution.

[0038] Figure 9 GO enrichment analysis of differentially expressed genes in rapeseed roots after different treatments following chitosan aqueous solution root irrigation (a: ATR; b: CTS; c: ATR+CTS). The rich factor is the ratio of the number of DEGs enriched in a specific GO entry to the total number of genes annotated in that entry. The higher the value, the greater the enrichment.

[0039] Figure 10 The KEGG enrichment factor map of DEGs in rapeseed roots after different treatments of chitosan aqueous solution root irrigation (a: ATR; b: CTS; c: ATR+CTS); where, the rich factor refers to the ratio of the number of DEGs annotated to a specific KEGG metabolic pathway to the total number of genes annotated to that pathway, and the larger the value, the higher the enrichment degree.

[0040] Figure 11 The main transcription factors of differentially expressed genes in different treatments compared to the control group after root irrigation with chitosan aqueous solution (A: ATR; B: CTS; C: ATR+CTS);

[0041] Figure 12 RT-qPCR validation diagrams of differentially expressed genes under different treatments after root irrigation with chitosan aqueous solution (A: ATR; B: ATR+CTS);

[0042] Figure 13 A total graph showing the differentially expressed genes in different metabolic processes under different treatments of chitosan aqueous solution root irrigation (A: ATR vs CK; B: ATR+CTS vs CK; C: ATR+CTS vs ATR);

[0043] Figure 14 The effect of chitosan aqueous solution on the physiological indicators of soil-grown rapeseed plants under spray application conditions (A: fresh weight, B: plant height);

[0044] Figure 15 The graph shows the effect of chitosan aqueous solution on the growth of soil-cultured rapeseed plants under spray application conditions (ATR represents application of water once at 5d, 10d, and 15d; T1 represents application of 10mg / kg CTS once at 5d, and application of water once at 10d and 15d; T2 represents application of 10mg / kg CTS once at 5d and 10d, and application of water once at 15d; T3 represents application of 10mg / kg CTS once at 5d, 10d, and 15d). Detailed Implementation

[0045] The technical solutions of this application will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0046] The rapeseed seeds and plant culture conditions used in the following examples are as follows:

[0047] Rapeseed: Sichuan Oil No. 45 (seeds purchased online). Plump rapeseed seeds without mold or damage were disinfected with a 5% sodium hypochlorite solution for 15 minutes. The seeds were then rinsed with pure water to remove the sodium hypochlorite. The seeds were then wrapped in damp gauze and placed in a seedling tray filled with shallow water. The trays were placed in a dark 30℃ incubator for one day. Nutrient solution was added to a depth of about 10cm in the seedling trays. A 1cm layer of washed and dried perlite was placed on top of the nutrient solution. Germinated rapeseed seeds were planted on the perlite, ensuring the roots were in contact with the nutrient solution. The seedling trays were placed in an artificial climate incubator with the following conditions: temperature 25℃, light-dark cycle (16h:8h), and light intensity 250μmol / m². 2 / s, relative humidity maintained at 70%, cultured for 6-8 days for later use. For soil-cultured experiments, seedlings cultured for 8 days were transferred to new containers and cultured for another 10 days before use.

[0048] Chitosan: Carboxylated chitosan (CTS, degree of carboxylation ≥80%, CAS No.: 9012-76-4), Shanghai Yuanye Biotechnology Co., Ltd.

[0049] The chemical composition of the nutrient solution is shown in Table 1:

[0050] Table 1

[0051]

[0052] Experimental soil

[0053] The test soil was collected from the topsoil of a field at a depth of approximately 20 cm in Lüliang City, Shanxi Province. After collection, it was sieved through a 2 mm sieve and placed in a ventilated area, avoiding direct sunlight. The physicochemical properties of the test soil were tested by Zhejiang Jiuan Testing Technology Co., Ltd., and the test results are shown in Table 2.

[0054] Table 2

[0055]

[0056] Detection of atrazine in plants

[0057] Weigh 0.5 g of rapeseed tissue into a 50 mL centrifuge tube, add 4 mL of ultrapure water and 5 mL of ethyl acetate, and homogenize for 2 min to fully disrupt the tissue. Place the centrifuge tube on a shaker and shake for 10 min, then sonicate at 100 Hz for 20 min. Weigh 1 g of NaCl and 4 g of anhydrous MgSO4, add them to the centrifuge tube, and vortex for 1 min to mix. Centrifuge at 7000 rpm / min for 3 min. Pre-load a purification solution (10 mg GCB + 50 mg PSA + 150 mg MgSO4) into a 2 mL centrifuge tube. Transfer 1.5 mL of the supernatant from centrifugation to the tube containing the purification solution, vortex for 1 min, and centrifuge at 7000 rpm / min for 3 min. Filter the supernatant through a 0.22 μm organic filter membrane for GC-MS analysis.

[0058] GC-MS conditions

[0059] GC conditions:

[0060] Chromatographic column: HP-5MS (0.25mm×30m, 0.25μm);

[0061] Helium flow rate: 1.2 mL / min;

[0062] Temperature program: 100℃, 20℃ / min to 230℃ and hold for 3 min, 10℃ / min to 300℃ and hold for 2 min;

[0063] Mass spectrometry conditions: Electron impact ionization (EI) was used, and the sample was ionized at an ion source temperature of 250 °C. The results were compared with standard spectra in the NIST database (National Institute of Standards and Technology, USA).

[0064] Mode: SIM mode;

[0065] Quantitative ion count: 200 (m / z);

[0066] Qualitative ions: 215 (m / z), 202 (m / z);

[0067] Peak emission time: 6.2 min

[0068] The formula for calculating atrazine residue is as follows:

[0069]

[0070] Where: X is the concentration of atrazine in the sample to be tested (mg / L); Ax is the peak area of ​​atrazine in the sample; A0 is the peak area of ​​atrazine standard sample; Vx is the sample volume (mL); V0 is the final volume (mL); Cs is the concentration of atrazine standard sample (mg / L).

[0071] Example 1

[0072] The promoting effect of chitosan on atrazine metabolism in nutrient-cultured rapeseed

[0073] (1) The promoting effect of chitosan hydroponic treatment on atrazine metabolism in rapeseed

[0074] Rapeseed seedlings with uniform growth were transplanted into nutrient solutions containing atrazine (0.02 mg / kg) and those without pesticides. Different concentrations of chitosan aqueous solution were added to the nutrient solutions to the corresponding concentrations. This experiment set up five treatment groups, with three replicates for each treatment: a control group (CK) without pesticides, a pesticide group (ATR), a pesticide + 1 mg / kg chitosan group (1 ppm), a pesticide + 10 mg / kg chitosan group (10 ppm), and a pesticide + 100 mg / kg chitosan group (100 ppm). Atrazine residues and rapeseed physiological indicators were measured in the plants at 2, 5, and 10 days.

[0075] The results are as follows Figure 1 As shown, the addition of CTS to the culture medium slowed the effect of atrazine on the fresh weight of rapeseed. After 10 days of treatment with 1, 10, and 100 mg / kg, the fresh weight of the CTS-treated groups was 75.96%, 78.99%, and 79.41% of that of the control group, respectively, while the fresh weight of the atrazine-only treatment group was 56.80% of that of the control group. The plant height of the CTS-treated groups was 85.15%, 85.23%, and 89.11% of that of the control group, respectively, while the plant height of the atrazine-only treatment group was 59.9% of that of the control group. This indicates that chitosan treatment significantly alleviated the effect of atrazine on the fresh weight and plant height of rapeseed. The residues in the CTS-treated groups were 84.17%, 69.07%, and 61.92% of those in the control group, respectively, indicating that the presence of CTS can significantly accelerate the metabolism of atrazine in rapeseed.

[0076] (2) The promoting effect of chitosan spray treatment on atrazine metabolism in rapeseed

[0077] Rapeseed seedlings with uniform growth were transplanted into nutrient solutions containing atrazine (0.02 mg / kg) and those without pesticides. Foliar spraying with chitosan aqueous solution of different concentrations was performed. This experiment set up five treatment groups, with three replicates for each treatment: control group (CK), pesticide group (ATR), pesticide + 1 mg / kg chitosan group (1 ppm), pesticide + 10 mg / kg chitosan group (10 ppm), and pesticide + 100 mg / kg chitosan group (100 ppm). Atrazine residues in the plants and rapeseed physiological indicators were measured at 2, 5, and 10 days.

[0078] The results are as follows Figure 2 As shown, after 10 days of treatment with 1, 10, and 100 mg / kg, the fresh weight of the CTS-treated groups was 74.38%, 72.53%, and 75.15% of that of the control group, respectively, while the fresh weight of the atrazine-only treatment group was 46.76% of that of the control group. The plant height of the CTS-treated groups was 91.96%, 92.85%, and 94.61% of that of the control group, respectively, while the plant height of the atrazine-only treatment group was 72.32% of that of the control group. This indicates that CTS treatment significantly alleviated the effects of atrazine on the fresh weight and plant height of rapeseed. The residues in the CTS-treated groups were 85.37%, 84.12%, and 83.99% of those in the control group, respectively, indicating that the presence of CTS can significantly accelerate the metabolism of atrazine in rapeseed.

[0079] Example 2: Verification of the effect of chitosan

[0080] Rapeseed seedlings were transferred to a nutrient solution containing atrazine (0.02 mg / kg) and cultured for three days under the same conditions as described above. After washing and drying the roots, the seedlings were transferred to a nutrient solution without atrazine. Then, hydroponic treatment was performed by adding chitosan stock solution dissolved in pure water to concentrations of 1 mg / kg, 10 mg / kg, and 100 mg / kg. A control group (CK) without chitosan was also included, with three replicates per group. Plant samples were collected on days 0, 2, 5, and 10 to determine atrazine residues in the plants. Results are as follows: Figure 3 As shown, the addition of CTS to the culture medium accelerated atrazine metabolism and reduced atrazine residues in rapeseed plants. After 2 days of treatment with 1, 10, and 100 mg / kg, the atrazine concentrations were 48.51%, 44.49%, and 39.58% of the control group, respectively, with no significant effect between different CTS concentrations at this point. After 10 days, the concentrations were 66.67%, 50.0%, and 33.34% of the control group, showing a concentration-dependent effect. The experiment demonstrates that CTS plays a crucial role in accelerating atrazine metabolism in rapeseed.

[0081] Example 3: Possible mechanism by which chitosan promotes atrazine metabolism in rapeseed plants

[0082] The following treatments were performed in a system of 140 ml nutrient solution and 2 rapeseed seedlings: nutrient solution blank group (CK), 0.02 mg / L atrazine treatment group (ATR), 10 mg / L chitosan treatment group (CTS), and atrazine + chitosan treatment group (ATR+CTS). Each treatment was set up in triplicate. Samples were collected at 2, 5, and 10 days of rapeseed growth for various index measurements, including relevant metabolic enzyme activities, transcriptome sequencing, and qPCR verification.

[0083] (1) Effects of chitosan hydroponic treatment on enzyme activity in rapeseed plants

[0084] Cytochrome P450 enzymes play a crucial role in phase I pesticide metabolism in plants, representing an important process for plants to cope with external stresses. Figure 4 As shown in Figure A, compared with the control group, the P450 enzyme activity significantly increased after 2 days of atrazine treatment; while after CTS treatment, the P450 enzyme activity did not change significantly compared with CK; however, after ATR+CTS mixed treatment, the P450 enzyme activity significantly increased at 2 days, and then returned to the same level as CK after 5 days. These results indicate that rapeseed regulates cell function and promotes atrazine metabolism by increasing P450 enzyme activity, and ATR+CTS further enhances P450 enzyme activity to promote atrazine metabolism.

[0085] Glutathione S-transferase (GST) is one of the important enzymes in phase II metabolism in plants. Figure 4 As known from the study, compared with the control group, the GST enzyme activity significantly increased after 0-5 days of atrazine addition. Although the enzyme activity decreased slightly at 10 days compared with 5 days, it was still higher than that of the control group. After CTS treatment, the GST enzyme activity did not change significantly compared with the control group. However, after ATR+CTS mixed treatment, the GST enzyme activity significantly increased from 0-5 days and was higher than that of the ATR-only treatment group, indicating that ATR+CTS further enhanced the GST enzyme activity and promoted the metabolism of atrazine. After 10 days, the activity decreased and was lower than that of the ATR-treated group. This may be because the atrazine content in the plant is low, and the plant itself does not need to increase the GST enzyme activity to accelerate metabolic regulation.

[0086] Glycosyltransferases (GTs) are among the important enzymes in plant dual metabolism. Figure 4 According to the findings of the Chinese Center for Disease Control and Prevention (CDC), compared with the control group, the activity of GTs enzymes increased slowly during the period of atrazine addition from 0 to 10 days, showing a significant difference from the control group at 10 days. After CTS treatment, the activity of GTs enzymes did not change significantly compared with the control group. However, after ATR+CTS combined treatment, the activity of GTs enzymes increased significantly from 0 to 5 days and was higher than that of the ATR-only treatment group, indicating that ATR+CTS further enhanced the activity of GST enzymes and promoted the metabolism of atrazine.

[0087] Superoxide dismutase (SOD) plays an important role in scavenging reactive oxygen species in plants. Figure 4 As shown in Figure D, compared with the control group, the SOD enzyme activity of the atrazine treatment group was higher, while the SOD enzyme activity of the ATR+CTS mixed treatment group was higher than that of the atrazine-only treatment group, indicating that ATR+CTS further enhanced the SOD enzyme activity to maintain the homeostasis of the plant.

[0088] Peroxidase (POD), by Figure 4 According to the findings in the study, compared with the control group, the POD enzyme activity significantly increased after 0-5 days of atrazine addition. Although the enzyme activity decreased slightly at 10 days compared with 5 days, it was still higher than that of the control group. After CTS treatment, the POD enzyme activity did not change significantly compared with CK. However, after ATR+CTS mixed treatment, the POD enzyme activity significantly increased from 0-5 days and was higher than that of the ATR-only treatment group, indicating that ATR+CTS further enhanced the POD enzyme activity and promoted the metabolism of atrazine. The activity decreased after 10 days, which may be because the atrazine content in the plant was low, and the plant itself did not need to increase the POD enzyme activity to accelerate metabolic regulation.

[0089] Glutathione reductase (GR), by Figure 4 As far as we know, compared with the control group, the activity of GR enzyme significantly increased after 0-5 days of atrazine addition. Although the enzyme activity decreased slightly at 10 days compared with 5 days, it was still higher than that of the control group. After CTS treatment, the GR enzyme activity did not change significantly compared with CK. However, after ATR+CTS mixed treatment, the GR enzyme activity increased significantly from 0-5 days and was higher than that of the ATR-only treatment group, indicating that ATR+CTS further enhanced the GR enzyme activity and promoted the metabolism of atrazine. After 10 days, the activity decreased and was lower than that of the ATR-treated group, which was similar to the change in GST. This may be because the atrazine content in the plant is low, and the plant itself does not need to increase the GR enzyme activity to accelerate metabolic regulation.

[0090] (2) Effects of chitosan spray treatment on enzyme activity in rapeseed plants

[0091] Cytochrome P450 enzymes play a crucial role in phase I pesticide metabolism in plants, representing an important process for plants to cope with external stresses. Figure 5As shown in Figure A, compared with the control group, the P450 enzyme activity significantly increased after 2 days of atrazine addition, but returned to the same level as CK after 5 days. However, after CTS treatment, the P450 enzyme activity did not change significantly compared with CK. Nevertheless, after ATR+CTS combined treatment, the P450 enzyme activity significantly increased at 2 days and remained higher than CK after 5 days. These results indicate that rapeseed regulates cell function and promotes atrazine metabolism by increasing P450 enzyme activity, and ATR+CTS further enhances P450 enzyme activity, thus promoting atrazine metabolism.

[0092] Glutathione S-transferase (GST) is one of the important enzymes in phase II metabolism in plants. Figure 5 As known from the study, compared with the control group, the GST enzyme activity significantly increased after 0-5 days of atrazine addition. Although the enzyme activity decreased slightly at 10 days compared with 5 days, it was still higher than that of the control group. After CTS treatment, the GST enzyme activity did not change significantly compared with the control group. However, after ATR+CTS mixed treatment, the GST enzyme activity significantly increased from 0-5 days and was higher than that of the ATR-only treatment group, indicating that ATR+CTS further enhanced the GST enzyme activity and promoted the metabolism of atrazine. After 10 days, the activity decreased and was lower than that of the ATR-treated group. This may be because the atrazine content in the plant is low, and the plant itself does not need to increase the GST enzyme activity to accelerate metabolic regulation.

[0093] Glycosyltransferases (GTs) are among the important enzymes in plant dual metabolism. Figure 5 According to the findings of the Chinese Center for Disease Control and Prevention (CDC), compared with the control group, the activity of GTs enzymes increased slowly during the period of atrazine addition from 0 to 10 days, showing a significant difference from the control group at 10 days. After CTS treatment, the activity of GTs enzymes did not change significantly compared with the control group. However, after ATR+CTS combined treatment, the activity of GTs enzymes increased significantly from 0 to 5 days and was higher than that of the ATR-only treatment group, indicating that ATR+CTS further enhanced the activity of GST enzymes and promoted the metabolism of atrazine.

[0094] Superoxide dismutase (SOD) plays an important role in scavenging reactive oxygen species in plants. Figure 5 As shown in Figure D, from 0 to 10 days, the SOD enzyme activity of the atrazine treatment group was higher than that of the control group, while the SOD enzyme activity of the ATR+CTS mixed treatment group was higher than that of the atrazine-only treatment group, indicating that ATR+CTS further enhanced SOD enzyme activity to maintain the homeostasis of the plant.

[0095] Peroxidase (POD), by Figure 5According to the findings in the study, compared with the control group, the POD enzyme activity significantly increased after 0-5 days of atrazine addition. Although the enzyme activity decreased slightly at 10 days compared with 5 days, it was still higher than that of the control group. After CTS treatment, the POD enzyme activity did not change significantly compared with CK. However, after ATR+CTS mixed treatment, the POD enzyme activity significantly increased from 0-5 days and was higher than that of the ATR-only treatment group, indicating that ATR+CTS further enhanced the POD enzyme activity and promoted the metabolism of atrazine. The activity decreased after 10 days, which may be because the atrazine content in the plant was low, and the plant itself did not need to increase the POD enzyme activity to accelerate metabolic regulation.

[0096] Glutathione reductase (GR), by Figure 5 As far as we know, compared with the control group, the GR enzyme activity slowly increased after 0-10 days of atrazine addition, and although the enzyme activity was higher than CK at 10 days; after CTS treatment, the GR enzyme activity did not change significantly compared with CK; however, after ATR+CTS mixed treatment, the GR enzyme activity increased significantly from 0-5 days and was higher than the ATR single treatment group, indicating that ATR+CTS further enhanced the GR enzyme activity and promoted the metabolism of atrazine; after 10 days, it declined and was lower than the ATR treatment group, and the change was similar to GST, which may be because the atrazine content in the plant is low, and the plant itself does not need to increase the GR enzyme activity to accelerate metabolic regulation.

[0097] (3) Transcriptome sequencing results

[0098] Transcriptome sequencing was performed in four treatment groups: CK (blank nutrient solution), ATR (atrazine treatment), CTS (chitosan treatment), and ATR+CTS (mixed atrazine and chitosan treatment). Each group had four biological replicates to explore the molecular mechanisms by which atrazine and chitosan promote atrazine metabolism in rapeseed.

[0099] Depend on Figure 6 It can be seen that differentially expressed genes (DEGs) are obtained by comparing different treatment groups with the control group (CK). For example... Figure 6 As shown in Figure A, compared with the CK group, the CTS group had a total of 2670 DEGs in rapeseed roots, of which 746 were upregulated and 1924 were downregulated; compared with the CK group, as shown in Figure A. Figure 6 As shown in Figure B, the ATR group treated rapeseed roots contained a total of 3324 DEGs, of which 784 were upregulated and 2540 were downregulated; Figure 6 As shown in Figure C, compared with the CK group, the ATR+CTS group had a total of 5889 DEGs in rapeseed roots, of which 1937 were upregulated and 3952 were downregulated.

[0100] The overlapping DEGs in the three processing groups (ATR, CTS, ATR+CTS) are as follows: Figure 7 As shown, the ATR, CTS, and ATR+CTS treatments contained 832, 629, and 2938 DEGs in the rapeseed roots, respectively, accounting for 25.03%, 23.56%, and 49.89% of the total DEGs. ATR and CTS together contained 138 DEGs, ATR and ATR+CTS together contained 1048 DEGs, and CTS and ATR+CTS together contained 597 DEGs. The three groups contained a total of 1306 DEGs, indicating that different treatments of rapeseed resulted in both common and unique DEGs in the roots.

[0101] Cytochrome P450, GST, GTS, POD, and ABC transporters are common catalytic enzymes in the phases I, II, and III metabolism of pesticides in plants. Therefore, this experiment aimed to identify DEGs related to cytochrome P450, GST, GTS, POD, and ABC transporters from both ATR alone and ATR+CTS mixed treatments, in order to pinpoint key regulatory genes in atrazine metabolism. Figure 8 As shown.

[0102] like Figure 8 There are 22 common genes encoding cytochrome P450 enzymes, of which 12 DEGs are upregulated and 10 are downregulated. The number of upregulated genes is greater than the number of downregulated genes. Among them, the genes LOC106357253 and LOC106357264, which are significantly upregulated, show ATR+CTS upregulation by 3.87-fold and 4.14-fold, respectively, corresponding to ATR upregulation of 3.5-fold and 2.33-fold. This indicates that atrazine induces the upregulation of P450-encoding genes in rapeseed roots, leading to enhanced cytochrome P450 enzyme activity and thus accelerating atrazine metabolism. The addition of CTS can enhance this upregulation effect, which is consistent with the results of cytochrome P450 enzyme activity measurement.

[0103] There are 22 common genes encoding glycosyltransferases, of which 14 DEGs are upregulated and 8 are downregulated. The number of upregulated genes is greater than the number of downregulated genes. Among them, the genes LOC106430366 and LOC111211449, which are significantly upregulated, show ATR+CTS upregulation by 4.01-fold and 3.7-fold, respectively, corresponding to ATR upregulation of 3.71-fold and 2.82-fold. This indicates that atrazine induces the upregulation of GTs encoding genes in rapeseed roots, leading to enhanced glycosyltransferase activity and thus accelerating atrazine metabolism. The addition of CTS can enhance this upregulation effect, which is consistent with the results of glycosyltransferase activity measurement.

[0104] There are 11 common genes encoding glutathione S-transferase enzymes, of which 2 DEGs are upregulated and 9 DEGs are downregulated. The number of upregulated DEGs is less than that of downregulated DEGs. However, in the ATR and ATR+CTS groups, the GST enzyme in rapeseed was activated, which may be due to gene redundancy caused by multiple genes co-encoding.

[0105] There are 13 common genes encoding peroxidase, of which 9 DEGs are upregulated and 4 are downregulated. The number of upregulated genes is greater than the number of downregulated genes. Among them, the genes LOC106402880 and LOC106451401, which are significantly upregulated, show ATR+CTS upregulation by 2.16-fold and 1.89-fold, respectively, corresponding to ATR upregulation of 2.12-fold and 1.55-fold. This indicates that atrazine induces the upregulation of POD-encoding genes in rapeseed roots, leading to enhanced peroxidase activity and thus accelerating atrazine metabolism. The addition of CTS can enhance this upregulation effect, which is consistent with the results of peroxidase activity measurement.

[0106] There are 10 common genes encoding ABC transporter proteins. Among them, 6 DEGs are upregulated and 4 are downregulated. The number of upregulated genes is greater than the number of downregulated genes. Among them, the genes LOC106377488 and LOC106439741 are significantly upregulated by ATR+CTS by 2.30-fold and 3.03-fold, respectively, corresponding to an upregulation of 1.99-fold and 3.02-fold in the ATR group. This indicates that atrazine induces the upregulation of genes encoding ABC transporter proteins in rapeseed roots, leading to enhanced ABC transporter protein activity and thus accelerating the transport of atrazine from the cell to the extracellular space. The addition of CTS can enhance this upregulation effect.

[0107] In conclusion, chitosan may have promoted the metabolism of atrazine and mitigated its stress effects by enhancing the upregulation of the coding genes for cytochrome P450, glycosyltransferase, peroxidase, and ABC transporter in rapeseed roots.

[0108] All selected DEGs were annotated and enriched for GO function. The results showed that the 3324 DEGs in the ATR group were enriched into 2257 GO items, with 208 showing significant enrichment (P<0.05); the 3670 DEGs in the CTS group were enriched into 2267 GO items, with 151 showing significant enrichment (P<0.05); and the 5889 DEGs in the ATR+CTS group were enriched into 3124 GO items, with 563 showing significant enrichment (P<0.05). The top 10 dominant items were selected for GO function classification analysis in this study.

[0109] Significantly enriched GO entries included three main categories: cellular composition and components, molecular function, and biological processes. For example... Figure 9As shown, A represents the ATR treatment group, B the CTS treatment group, and C the ATR+CTS treatment group. The rich factor refers to the ratio of the number of DEGs enriched in a specific GO entry to the total number of genes annotated for that entry; the higher the value, the greater the enrichment. The GO entry common to all three treatments is the redox process in the BP category, indicating that both the exogenous substances atrazine and chitosan activated the rapeseed's own antioxidant system. The application of chitosan may promote atrazine metabolism by enhancing the induction of the antioxidant system.

[0110] Furthermore, ATR and ATR+CTS share significantly enriched GO entries including intrinsic component of membrane, integral component of membrane, membrane part, membrane, and catalytic activity, suggesting that chitosan may accelerate the degradation of atrazine by altering the composition and catalytic activity of the cell membrane.

[0111] Several treatment groups also had specific GO entries in their DEGs. For example, the DEGs of the CTS group were significantly enriched in GO entries such as DNA binding and metal ion binding (BP category), and regulation of biological process, regulation of cellular process, regulation of metabolic process, aromatic compound biosynthetic process, heterocycle biosynthetic process, and organic cyclic compound biosynthetic process (MF category). The DEGs of the ATR group were significantly enriched in GO entries such as localization, transport, establishment of localization, and oxidation-reduction process (BP category). The DEGs of the ATR+CTS group were significantly enriched in GO entries such as metabolic process and biological regulation (BP category), and ion binding (MF category). This indicates that different treatments produce specific stimuli to plants. For example, CTS mainly affects the biosynthesis of organic cyclic compounds and the decomposition of drugs by influencing the ability of roots to bind and the ability of cells to regulate. ATR promotes plant metabolism through transport and redox processes. ATR+CTS mainly accelerates atrazine metabolism by activating ion binding and regulating biological metabolic processes.

[0112] By comparing DEGs with the KEGG public database and performing pathway annotation and enrichment analysis, the regulated metabolic pathways in different treatments were identified. In the ATR group, DEGs from rapeseed roots were annotated into 113 pathways, of which 25 pathways were significantly enriched; in the CTS group, DEGs from rapeseed roots were annotated into 105 pathways, of which 18 pathways were significantly enriched; and in the ATR+CTS group, DEGs from rapeseed roots were annotated into 117 pathways, of which 25 pathways were significantly enriched. Enrichment factor plots were created for the top 10 significantly enriched pathways in each treatment group, as shown below. Figure 10Where A is the ATR treatment group, B is the CTS treatment group, and C is the ATR+CTS treatment group. The rich factor refers to the ratio of the number of DEGs enriched in a specific GO entry to the total number of genes annotated in that entry. The higher the value, the greater the enrichment.

[0113] Based on the number of enriched DEGs, the main metabolic pathways in the ATR group are plant hormone signal transduction, plant-pathogen interaction, and the MAPK signaling pathway. The most important enriched pathways are the degradation of valine, leucine, and isoleucine, and the biosynthesis of phenylpropane.

[0114] Based on the number of enriched DEGs, the main metabolic pathways in the CTS group are plant hormone signal transduction, phenylpropanoid biosynthesis, and plant-pathogen interaction. From the perspective of enriched factors, carotenoid synthesis and phenylpropanoid biosynthesis are the most important pathways.

[0115] Based on the number of enriched DEGs, the main metabolic pathways in the ATR+CTS group are plant hormone signal transduction, plant-pathogen interaction, and phenylpropane biosynthesis. In terms of enriched factors, nitrogen metabolism, glyoxylate and dicarboxylate metabolism are the most important pathways.

[0116] ATR and ATR+CTS share six metabolic pathways: plant hormone signal transduction, MAPK signaling pathway-plant, phenylpropane biosynthesis, cysteine ​​and methionine metabolism, glutathione metabolism, and plant-pathogen interaction. The combined group has a more significant activation effect.

[0117] It is speculated that chitosan promotes the synthesis of plant hormones ethylene and polyamines, activates the MAPK pathway to transmit stress signals, enhances the induction of the phenylpropane pathway, strengthens glutathione synthesis and metabolism, promotes the synthesis of secondary metabolites, and accelerates atrazine metabolism.

[0118] Transcription factors (TFs) are protein molecules that specifically bind to the upstream nucleotide sequences of target genes, regulating their transcription process. They are widely involved in plant growth and development and in responding to abiotic stresses. For example... Figure 11As shown, ATR vs CK(A) involves 51 transcription factor families, with the top five families having the most DEGs being BHLH, ERF, NAC, C2H2, and MYB-related. CTS vs CK(B) involves 54 transcription factor families, with the top five families having the most DEGs being ERF, BHLH, NAC, WRKY, and C2H2. ATR+CTS vs CK(C) involves 55 transcription factor families, with the top five families having the most DEGs being ERF, BHLH, NAC, WRKY, and MYB-related.

[0119] Atrazine alone and in combination with chitosan were selected for treatment. Both treatments share four transcription factor families. Compared with atrazine alone, the combination treatments resulted in enhanced upregulation of bHLH and ERF, NAC, and MYB-related transcription factor families. Among them, the MYB-related transcription factor family is a plant-specific family involved in the synthesis of secondary metabolites and cell morphology regulation; bHLH is involved in regulating the biosynthesis of flavonoids; the ERF family are ethylene-responsive factors; and the NAC transcription factor family plays a role in plant growth and development and responses to abiotic stresses.

[0120] Therefore, it is speculated that the process by which chitosan promotes atrazine metabolism involves signal transduction, ethylene activation, and the synthesis and accumulation of plant secondary metabolites such as flavonoids. The NAC family may also be involved in stress responses.

[0121] To verify the reliability of the transcriptome data, this experiment selected one gene from each of the P450, GST, GTS, POD, and ABC genes commonly found in both ATR and ATR+CTS treatments for RT-qPCR validation. The results are as follows: Figure 12 As shown in (A: ATR vs CK; B: ATR+CTS vs CK), the final RT-qPCR results showed a consistent up- and down-regulation trend with the transcriptome sequencing results, indicating that the transcriptome data is reliable.

[0122] To further clarify the role of differentially expressed genes in metabolic processes, this application used the Mapman program to import all significantly differentially expressed genes into Mapman, ultimately yielding... Figure 13 (A:ATR vs CK; B:ATR+CTSvs CK; C:ATR+CTS vs ATR), such as Figure 13As shown, the expression of genes related to the atrazine, ascorbic acid, and glutathione pathways was reduced, and a large number of genes were involved in secondary metabolism. Among them, genes involved in polypropylene, terpenes, phenylpropanoids, flavonoids, and phenolic compounds were upregulated, and the combined treatment of ATR+CTS further promoted the expression of these genes. Significant changes were also observed in genes involved in cell wall, lipid, one-carbon metabolism, and glucose metabolism, with both upregulated and downregulated genes.

[0123] Example 4

[0124] Chitosan's effect on mitigating pesticide damage in rapeseed cultivation in soil

[0125] The initial concentration of atrazine in the soil was set at 0.05 mg / kg. Approximately 200 g of soil was weighed into a beaker, and atrazine standard solution was added dropwise. After mixing, the mixture was placed in a fume hood for 2 hours to allow the organic solvent to evaporate. The air-dried soil was then mixed thoroughly with the remaining soil, and an appropriate amount of deionized water was added to bring the soil moisture content to 30% of its maximum. The mixture was then passed through a 2 mm sieve. The mixed soil was placed in a dark place at 25°C for 24 hours to equilibrate.

[0126] 300g of dry soil was divided into disposable plastic flowerpots. Healthy and uniformly growing rapeseed seedlings, whose roots had been cleaned and allowed to recover overnight in nutrient solution, were planted in the plastic flowerpots and placed back into an artificial climate incubator for medium-temperature cultivation. This experiment included four treatment groups, each with three replicates: pesticide treatment group (ATR), pesticide + one-time chitosan treatment group (T-1), pesticide + two-time chitosan treatment group (T-2), and pesticide + three-time chitosan treatment group (T-3). The chitosan concentration was 10mg / kg, applied by spraying. Treatment times were: first time: 5 days after transplanting; second time: 10 days after transplanting; third time: 15 days after transplanting. After the third spray, the plants were left to stand for another 15 days, after which plant height and fresh weight were measured and photographed.

[0127] 30 days later, if Figure 14 As shown in the figure, A represents the fresh weight results, and B represents the plant height results. The fresh weights of T1, T2, and T3 were 27.76%, 44.61%, and 89.85% of the CK, respectively, showing a clear concentration dependence. The fresh weight of the ATR treatment was 14.34% of the CK. The plant heights of T1, T2, and T3 were 72.48%, 84.04%, and 94.22% of the CK, respectively, showing a clear concentration dependence. The plant height of the ATR treatment was 50.74% of the CK.

[0128] Growth status as follows Figure 15 As shown, the rapeseed growth after applying chitosan aqueous spray was significantly closer to that of the control (CK).

[0129] The above experiments show that the chitosan aqueous solution of the present invention can effectively alleviate the phytotoxicity of atrazine to rapeseed under root irrigation or spraying conditions. Considering the cost of use, a chitosan aqueous solution of 10 ppm can achieve good results.

[0130] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. A method for reducing atrazine damage to plants, characterized in that, include: Chitosan was formulated into a chitosan aqueous solution, which was then applied to plants grown in atrazine-contaminated soil or nutrient solution; the plant was rapeseed; the structural formula of the chitosan was: ； The chitosan has a molar mass of 1526.5 g / mol and a molecular formula of C2. 56 H 103 N9O 39 ; The application method is irrigation or spraying; the concentration of the chitosan aqueous solution is 5~25mg / kg; the chitosan aqueous solution is applied 1~4 times, with the first application time being 4~6 days after the seedlings are transplanted into atrazine-contaminated soil or nutrient solution, and the interval between each application is 4~6 days.

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