Application of malic acid and preparation in relieving or improving inhibition of pesticide residues on soil

Abstract

The invention discloses application of malic acid as a soil remediation agent in relieving or improving inhibition of pesticide residues on soil, and belongs to the technical field of new application technologies of biological secretions, especially soil remediation. An enzyme activity-microbiological mechanism is deeply studied, and an in-situ zymography method is utilized to find that the application of malic acid can relieve anti-competitive inhibition caused by imidacloprid, reduce combination of imidacloprid and enzyme and recover enzyme activity. The malic acid and root exudates can reduce the pH value of the soil, and meanwhile the problem that the content of utilized phosphorus, the content of nitrate nitrogen and the content of organic carbon in the soil are reduced due to imidacloprid is effectively solved. The invention provides a feasible remediation technology for the pesticide-contaminated soil, and a new way is provided for healthy development of agriculture.

Description

Technical Field

[0001] The present invention belongs to the new application technology of malic acid in biological secretions, and particularly relates to the technical field of soil improvement and remediation, specifically to the application of malic acid and its preparations as soil remediation agents in alleviating or improving the inhibition of soil by pesticide residues. Background Art

[0002] As early as in the 1990s, China's apple production and planting area ranked first in the world, and it is a major apple-producing country. As an economic tree, apple trees can increase the income of local farmers and play ecological functions such as windbreak and sand fixation, air purification, prevention of soil erosion and water conservation. However, according to the "Chinese Fruit Tree Pests Record", there are more than 100 kinds of pests and diseases on apples, which has led to the frequent use of chemical pesticides.

[0003] Problems such as over-application and unscientific and unreasonable pesticide application exist in the process of using pesticides in orchards, resulting in high detection rates and high residues of pesticides in orchards. Moreover, it will have a certain impact on the transfer of pollutants and nutrient cycling in the soil-plant system by affecting soil physical and chemical properties, enzyme activities, and the evolution of microbial communities. The impact of pesticides on soil enzyme activities is related to the types of pesticides and soil enzymes, soil physical and chemical properties, and soil pesticide residue concentrations. At present, most of the research on soil pollution focuses on inorganic pollution, such as drought stress, heavy metal stress, salt stress, etc. There is still a lack of research on organic pollution, such as the analysis of pollution sources of organic pesticides left after application, the impact of pesticides on soil enzyme activities and microorganisms, and the mechanism of soil pollution remediation. As an important indicator for domestic and foreign scholars to evaluate soil environmental quality and safety, soil enzyme activity, combined with metagenomics widely used in recent years, can further explain the action mechanism of pollutants on soil. The current enzyme activity measurement methods used in research are still the traditional spectrophotometry and fluorescence analysis methods. However, the soil enzyme activities measured by these methods are only the maximum potential enzyme activities in the soil, and cannot accurately reflect the real situation of the distribution of soil enzyme activities in the environment. In-situ zymography is a two-dimensional distribution technology for analyzing soil enzyme activities by integrating fluorescent substrates recently (Spohn and Kuzyakov; 2013, Vandooren, et al.; 2013). This technology is very useful for various hydrolases, can provide a large amount of information on hydrolase activities, and reflect the form and localization of enzymes. Therefore, the further combination of in-situ zymography and metabolomics can effectively and intuitively explain the changes in soil ecology.

[0004] Imidacloprid (IMI), as a widely used neonicotinoid insecticide, is widely used in orchards due to its high efficiency in pest control. Imidacloprid residues can cause various stresses to plants, including oxidative stress , metabolic disorders and changes in the rhizosphere microbial community , these stress mechanisms not only affect the growth and development of plants, but may also pose potential risks to the health of ecosystems. Therefore, it is necessary to use them reasonably and study effective remediation and degradation measures for residual pesticides. Summary of the Invention

[0005] The object of the present invention is to study the enzyme activity-microbiology mechanism in depth by investigating the effects of imidacloprid on nutrient cycling, root characteristics, soil enzyme kinetics and rhizosphere microorganisms in the soil-plant system, and combining with the application of root exudates. It is found by in-situ zymography that the application of malic acid can relieve the uncompetitive inhibition caused by imidacloprid, reduce the binding of imidacloprid and the enzyme, and restore the enzyme activity. Malic acid and root exudates can reduce the soil pH value, and at the same time effectively improve the problems of reduced available phosphorus content, nitrate nitrogen content and organic carbon content in the soil caused by imidacloprid.

[0006] First, the present invention provides the application of malic acid in alleviating or improving the inhibition of soil by pesticide residues.

[0007] In the above application, preferably, the inhibition of soil by pesticide residues includes at least one of reduced soil enzyme activity, reduced phosphorus content, reduced nitrate nitrogen content and reduced organic carbon content. Among them, preferably, the soil enzyme is BG enzyme (β-glucosidase). The pesticide is imidacloprid. The preferred method of the application of the present invention is to slowly release malic acid into the soil with pesticide residues so as to establish an effective concentration gradient of malic acid in the soil, because if malic acid cannot react with the pesticide in time, its concentration will be too high and affect the soil, such as acidification; it can slowly release malic acid into the soil through a Rhizon sampler and culture it to establish a gradient in the soil around the entire Rhizon sampler (artificial root). The concentration of malic acid is appropriately adjusted according to the degree of pesticide residues, and generally can be 100-400 μmol C / mL. Preferably, malic acid with a concentration of 200 μmol C / mL can be added to the rhizobox at a rate of 1.0 mL per day through the Rhizon sampler (artificial root) and cultured for 14 days to ensure the establishment of a gradient around the entire artificial root.

[0008] Second, the present invention also provides the application of plant root exudates containing malic acid in alleviating or improving the inhibition of soil by pesticide residues. Among them, preferably, the inhibition of soil by pesticide residues includes at least one of reduced soil enzyme activity, reduced phosphorus content, reduced nitrate nitrogen content and reduced organic carbon content. The plants can be some fruit trees such as apple trees, pear trees, peach trees, etc. The soil enzyme is BG enzyme (β-glucosidase). The pesticide is imidacloprid.

[0009] Thirdly, the present invention also provides an application of a malic acid preparation in alleviating or improving the inhibition of soil by pesticide residues. Among them, the malic acid preparation may be a medicament added with malic acid, such as a composite preparation made by adding malic acid to some agricultural biological bacterial agents or bacterial fertilizers, such as biological bacterial agents, and then applied to the soil. The inhibition of the soil by pesticide residues includes at least one of a decrease in soil enzyme activity, a decrease in phosphorus content, a decrease in nitrate nitrogen content, and a decrease in organic carbon content. The soil enzyme is BG enzyme (β-glucosidase). The pesticide is imidacloprid.

[0010] The present invention has found through research that applying malic acid can relieve the uncompetitive inhibition caused by imidacloprid. Malic acid and imidacloprid are combined through hydrogen bonds and charge transfer, thereby reducing the binding of imidacloprid and the enzyme and restoring the enzyme activity. Malic acid and root exudates can reduce the soil pH value, and at the same time effectively improve the problems of the reduction of available phosphorus content, nitrate nitrogen content, and organic carbon content in the soil caused by imidacloprid. Therefore, it can be used as a soil remediator for imidacloprid pollution. The present invention provides a practical remediation technology for imidacloprid-polluted soil, providing new ideas and feasible ways for the green, sustainable and healthy development of agriculture. Description of the Drawings

[0011] Figure 1 It is a verification test diagram in a rhizobox. Among them, (a) is a real-time photo of the rhizobox after placing Rhizon® samples before the start of the experiment. The white line in the middle is the artificial root, and the dark shaded part is the soil sampling range, which is determined by the change range of enzyme activity in the zymogram; (b) is a zymogram taken under ultraviolet (UV) light with an excitation wavelength of 355 nm in a dark room; (c) is an example of a zymogram showing the response of malic acid to β-glucosidase activity under the action of imidacloprid. The right legend is proportional to the enzyme activity (nmolcm - 2 h -1 ).

[0012] Figure 2 Soil zymogram analysis diagram; among them, (a): The zymogram showing the response of malic acid to β-glucosidase activity under different treatments. After adding imidacloprid, the activity of BG enzyme decreased. (b): After adding malic acid, the activity of BG enzyme increased. (c), (d), (e): After simulating the secretion of malic acid by artificial roots, it was found that both the hot spot area of BG enzyme and the extension range of the enzyme increased. It shows that malic acid can repair the decrease in enzyme activity caused by imidacloprid residues.

[0013] Figure 3 It is a soil enzyme kinetic analysis diagram.

[0014] Figure 4 It is a content analysis diagram of imidacloprid and malic acid in the soil.

[0015] Figure 5 It is the Fourier transform infrared spectroscopy analysis diagram of rhizosphere soil.

[0016] Figure 6 It is the change diagram of soil-related parameters before and after adding malic acid. Among them, (a): the pH value of the soil under different treatments. After adding malic acid, the soil pH decreased. (b): the available phosphorus content in the soil under different treatments. After adding malic acid, the available phosphorus content in the soil increased. (c): the nitrate nitrogen content in the soil under different treatments. After adding malic acid, the nitrate nitrogen content in the soil increased significantly. (d): the change of dissolved organic carbon under different treatments. After adding malic acid, the dissolved organic carbon content in the soil increased significantly.

[0017] Figure 7 It is the analysis result diagram of apple root exudates extraction. Specific implementation mode

[0018] The following embodiments are further descriptions of the content of the present invention to explain the technical content of the present invention. However, the substantial content of the present invention is not limited to what is described in the following embodiments. Those of ordinary skill in the art can and should know that any simple changes or substitutions based on the substantial spirit of the present invention should belong to the protection scope required by the present invention.

[0019] Example 1

[0020] Experiment on the effect of malic acid on the enzyme activity at the root-soil interface polluted by the pesticide imidacloprid:

[0021] (1) Collect unpolluted soil

[0022] Before the experiment, soil samples were collected to measure the basic physical and chemical properties of the soil (organic matter, total nitrogen, nitrate ammonium nitrogen, available phosphorus, available potassium content, pH, etc.); the soil samples were collected from the surface layer (0-10 cm deep) of the Weibei Dryland Experimental Station of Northwest A&F University in Baishui County, Shaanxi Province, China in 2022 (east longitude 109°56′, north latitude 35°21′, altitude 838 m). To avoid the interference of malic acid and the use of pesticides, the soil was collected from fallow land 20 meters away from apple trees, and the land had not been planted with crops and fruit trees for more than 5 years. The soil was mixed evenly by hand, and at the same time, tree roots and stones were removed, and then the soil was put into a sealed bag and stored in an environment of minus 4°C. Before cultivation, the soil was passed through a 2-mm sieve. Before treating the soil, the concentration of imidacloprid remaining in the soil was measured, and the result was lower than the detection limit (0.01 mg / kg).

[0023] (2) Make a rhizobox

[0024] The whole experiment was carried out in a rhizobox (8×10×2 cm). The rhizobox is a transparent plastic box, and the front panel is detachable. When opened, it will not affect the soil surface and the artificial root (Rhizon) (Figure 1 ). A 100-mm-long Rhizon® sampler (Rhizosphere Research Products, Wageningen) was buried along the longitudinal center, with its upper surface at the same level as the soil. When loading the soil, the rhizobox was placed horizontally, one side was opened, and then sieved soil was slowly put in. After pouring a small amount of soil into the rhizobox, the opened side was closed, the sample was placed vertically again, and then gently shaken to make it uniform. The above steps were repeated until the rhizobox was filled with stable soil. Specifically, 1.2 kg of soil was evenly divided into three parts and sieved through a 2-mm sieve. Each soil sample was evenly transferred into two rhizoboxes. Then, imidacloprid (IM) solution was added dropwise to the soil surface with a pipette. In actual applications, the dosage of pesticides is often higher than the recommended dosage. To consider this situation, three imidacloprid concentrations were set in the rhizobox (8×10×2 cm): 0 mg / kg (IM0), 1.0 mg / kg (IM1, recommended concentration), and 5.0 mg / kg (IM5, 5 times the recommended concentration). The soil sample with a concentration of 0 mg / kg was treated with an equal amount of distilled water.

[0025] (3) Simulating the process of root exudation of malic acid into the soil

[0026] Using paired rhizoboxes with the same concentration of imidacloprid, the addition of malic acid (MA) or distilled water (CK) was compared through the Rhizon® sampler (artificial root). Malic acid (MA) was purchased from Yuanye Bio-Technology Co., Ltd., Shanghai, China. Four replicates were prepared for each treatment, and a total of 24 rhizoboxes were obtained for the experiment. The prepared rhizoboxes were first cultured for 6 days. On the 7th day, malic acid with a concentration of 200 μmol C / mL was added to the rhizobox at a rate of 1.0 mL per day through the Rhizon® sampler (artificial root). A 14-day culture was selected to ensure the establishment of a gradient around the entire artificial root. During the entire culture period, the rhizoboxes were kept in a climate chamber with a temperature stabilized at 25±1°C.

[0027] As Figure 1 For the verification and result figures, the white line in the middle of the rhizobox is the artificial root. Among them, (a) is a real-time photo of the rhizobox after placing the Rhizon® sample before the experiment. The dark shaded part is the soil sampling range, which is determined by the change range of enzyme activity in the zymogram; (b) is a zymogram taken in a dark room under ultraviolet (UV) light with an excitation wavelength of 355 nm; (c) is an example of a zymogram showing the response of β-glucosidase activity to malic acid under the action of imidacloprid. The right legend is proportional to the enzyme activity (nmol·cm -2 h -1); (d) Compared with the control, the soil enzyme activity decreased significantly on the third day after imidacloprid addition. On the seventh day, the difference was not obvious. On the thirtieth day, the soil enzyme activity with imidacloprid addition was not obvious compared with the control.

[0028] Soil enzyme spectrum analysis: Conducted after 28 days of cultivation. Soil enzyme spectrum analysis was used to determine the spatial distribution of soil enzymes around Rhizon® after the addition of malic acid in the presence of imidacloprid. As Figure 2 .

[0029] As Figure 3 , imidacloprid addition decreased the BG enzyme activity. After using the rhizoid to simulate the secretion of malic acid, it was found that both the hot spot area and the extension range of the BG enzyme increased. This indicates that malic acid can repair the decrease in enzyme activity caused by imidacloprid residues. Enzyme kinetic analysis showed that the application of imidacloprid led to a decrease in both Vmax (maximum reaction rate) and Km (Michaelis constant) of the soil, resulting in uncompetitive inhibition of the soil BG enzyme activity. After the application of malic acid, Vmax increased, Km increased, and the enzyme activity increased, which relieved the uncompetitive inhibition caused by imidacloprid to a certain extent. Malic acid may repair soil enzyme activity through the following mechanisms: (1) Competitive displacement: Malic acid may competitively bind to the enzyme-substrate complex with imidacloprid, thereby displacing imidacloprid and restoring enzyme activity. (2) Hydrogen bond and charge transfer: Since malic acid has carboxyl groups and can bind to imidacloprid through hydrogen bonds and charge transfer, less imidacloprid binds to the enzyme-substrate complex, restoring enzyme activity. To verify this idea, we also measured the contents of imidacloprid and malic acid in the soil, and the results were as Figure 4 . In the soil with malic acid addition, the concentrations of both imidacloprid and malic acid decreased, indicating that malic acid and imidacloprid are bound through hydrogen bonds and charge transfer, thereby reducing the binding of imidacloprid and enzymes and restoring enzyme activity.

[0030] As Figure 5 , further Fourier transform infrared spectroscopy analysis of rhizosphere soil showed that the characteristic absorption peaks in the Fourier transform infrared spectrum (FTIR) changed at approximately 1700 - 1730 cm⁻¹ after the application of malic acid, indicating the C=O stretching vibration of the carboxyl group. This shows that the carboxyl group (–COOH) and hydroxyl group (–OH) in malic acid can indeed form coordination bonds or hydrogen bonds with the nitrogen atoms in imidacloprid, which is also the fundamental reason why malic acid can repair imidacloprid-contaminated soil.

[0031] Figure 6It is a graph showing the changes in soil-related parameters before and after adding malic acid. Among them, (a): The pH value of the soil under different treatments. After adding malic acid, the soil pH decreases. (b): The available phosphorus content in the soil under different treatments. After adding malic acid, the available phosphorus content in the soil increases. (c): The nitrate nitrogen content in the soil under different treatments. After adding malic acid, the nitrate nitrogen content in the soil increases significantly. (d): The change in dissolved organic carbon under different treatments. After adding malic acid, the dissolved organic carbon content in the soil increases significantly. Different lowercase letters indicate significant differences, and the differences decrease in sequence from a with the letters, and the difference of d is not obvious.

[0032] Example 2

[0033] Extraction and analysis of root exudates

[0034] (1)Collection of soil basic physical and chemical property samples

[0035] Soil samples were collected before the experiment to determine the basic physical and chemical properties of the soil (organic matter, total nitrogen, nitrate ammonium nitrogen, available phosphorus, available potassium content, pH, etc.).

[0036] (2)Extraction and analysis of apple root exudates

[0037] The in situ collection method was used to collect root exudates. Three apple trees with similar breast diameter and size were selected for root exudates collection at the Weibei Dryland Experimental Station of Northwest Agriculture and Forestry University in Baishui County, Shaanxi Province, China (109°56′ east longitude, 35°21′ north latitude, 838 m above sea level). The fine roots were collected mainly by looking for the 0-10 cm soil layer buried in the surface layer along the exposed roots of each selected tree, and they were completely dug out. The soil particles on the surface of the fine roots were rinsed with deionized water, and other polymers on the surface were removed with tweezers; then the clean and complete roots were placed in a sterile 50 ml syringe, filled with about 1 mm sterile quartz sand, and then sealed with a syringe rubber stopper. The sterile syringe containing the selected fine roots was wrapped with tin foil and buried back in the original place (following the original growth direction of the root system, and fixing the syringe with the original soil to restore the original growth environment of the root system as much as possible); after a stabilization period of about 2 days, 20 ml of carbon-free nutrient solution (mainly containing 0.5 mM NH4NO3, 0.1 mM KH2PO4, 0.2 mM K2SO4, 0.2 mM MgSO4, 0.3 mM CaCl2) prepared in advance was added to each sterile syringe to eliminate the interference of soluble carbon, and a control was set up in each sample plot. After 24 hours, use a sterile syringe with a 0.22 μm filter to collect the nutrient solution in each syringe containing root secretions, and store the collected solution in a cold storage in time. Use a 0.22 μm syringe filter for filtration, and ensure that the filtration is completed within 24 hours after collection to obtain apple root secretions.

[0038] Using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) analysis, we found that among a total of 8743 mass features detected by positive (51.3%) or negative ions (48.7%), the reported features using the database (http: / / www.mzcloud.org) (Korenblum et al., 2020, Wright-Muelas et al.; 2020) included fatty acyl groups, carboxylic acids and their derivatives, organic oxygen compounds, steroids and steroid derivatives, organic nitrogen compounds, phenols, benzene and substituted derivatives, prenol lipids, pyridines and their derivatives, isoflavones, organophosphates and their derivatives, and purine nucleotides. Figure 7In Figure a, principal component analysis (PCA) showed that, according to the imidacloprid (IM) addition amount, the annotated metabolites strongly clustered, and PC 1 and PC 2 accounted for 43.28% and 25.77% of the variance, respectively (pANOSIM < 0.05) (Figure a). Compared with the control group CK, a total of 56 differential metabolic features were regulated in the IM group, including 22 down-regulated genes and 34 up-regulated genes (log 2 (fold change) > 1.5, q < 0.05, Figure b). Using a 2-fold change threshold, we found that 27 metabolites were significantly different in the IM addition compared with CK (p < 0.05, log 2 [fold change] > |1|). In particular, compared with CK, L-malic acid and L-2-hydroxyglutaric acid were specifically enriched in the IM additive (fold change > 3) (Figure c), which is related to the tricarboxylic acid cycle (TCA cycle). Therefore, the addition of IM (imidacloprid) enriched specific organic acids, namely L-malic acid and related product L-2-hydroxyglutaric acid, in the apple rhizosphere, and the malic acid content was the highest. Using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, we found that the tricarboxylic acid cycle, carbon metabolism, glyoxylate and dicarboxylate metabolism, biosynthesis of secondary metabolites, lysine degradation, sulfur metabolism, biosynthesis of amino acids, alanine, aspartate and glutamate metabolism, pyruvate metabolism, and carbon fixation in photosynthetic organisms were ranked among the top 10 KEGG pathways, and the KEGG pathways in the IM additive were enriched compared with CK (p < 0.05, t-test, Figure d). We focused on the TCA cycle and the biosynthesis pathway of secondary metabolites because they had high enrichment factors and P values respectively.

[0039] The present invention discovers that apple root exudates, especially malic acid, can relieve the uncompetitive inhibition caused by imidacloprid, restore the activity of soil BG enzyme, reduce the soil pH value, and at the same time effectively improve the problems of reduced available phosphorus content, nitrate nitrogen content, and organic carbon content in the soil caused by imidacloprid. Therefore, the present invention provides a practical technology for repairing pesticide-polluted soil, providing new ideas and feasible ways for the green, sustainable and healthy development of agriculture.

[0040] It should be noted that the above technical content of the present invention is only for explaining and clarifying the technical essence of the present invention to enable those skilled in the art to understand it, so the technical content is not used to limit the actual protection scope of the present invention. The actual protection scope of the present invention shall be subject to what is described in the claims. Those skilled in the art should know that any modifications, equivalent substitutions, and improvements made based on the essential spirit of the present invention shall be within the actual protection scope of the present invention.

Claims

1. Application of malic acid in alleviating or improving inhibition of soil by pesticide residues.

2. The application according to claim 1, characterized in that, The inhibition of soil by pesticide residues includes at least one of decreased soil enzyme activity, decreased phosphorus content, decreased nitrate nitrogen content, and decreased organic carbon content, and the soil enzyme is BG enzyme.

3. The application according to claim 1, wherein The pesticide is imidacloprid.

4. The application according to any one of claims 1-3, characterized in that, Slowly release malic acid into the soil through a Rhizon sampler to establish a gradient around the Rhizon sampler.

5. Application of plant root exudates containing malic acid in alleviating or improving inhibition of soil by pesticide residues.

6. The application according to claim 5, characterized in that, The inhibition of soil by pesticide residues includes at least one of decreased soil enzyme activity, decreased phosphorus content, decreased nitrate nitrogen content, and decreased organic carbon content, and the soil enzyme is BG enzyme.

7. The application according to claim 5, characterized in that The pesticide is imidacloprid.

8. Application of malic acid preparation in alleviating or improving inhibition of soil by pesticide residues.

9. The application according to claim 8, characterized in that, The inhibition of soil by pesticide residues includes at least one of decreased soil enzyme activity, decreased phosphorus content, decreased nitrate nitrogen content, and decreased organic carbon content, and the soil enzyme is BG enzyme.

10. The application according to claim 8, wherein The pesticide is imidacloprid.

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