A method for reducing soil-plant residues of compound fungicides by combining nanomaterials and nitrification inhibitors

Abstract

This invention relates to the field of soil treatment technology, specifically a method and its application for reducing compound fungicides in soil-plant systems by combining nano-silica and nitrification inhibitors. Nitrification inhibitors are used as a nitrogen management measure, and the application of nanomaterials influences soil abiotic properties and bacterial communities to drive the degradation of paclobutrazol and carbendazim. The application of nitrification inhibitors (DCD) produces multiple effects: significantly reducing carbendazim residues in soil-plant systems, paclobutrazol residues in soil, and NO3 in plants. ‑ -N and promote the content of soluble sugars and proline in plants. DCD reduces the activity of the soil electron transport system and β-glucosidase activity, while the addition of nano-SiO2 can mitigate this negative effect. Nano-SiO2 selectively enhances the performance of DCD in reducing compound fungicide residues in pak choi and reducing paclobutrazol residues, but hinders the degradation of carbendazim in pak choi.

Description

Technical Field

[0001] This invention relates to the field of soil treatment technology, specifically a method and its application for reducing the effects of combined fungicides on soil-plant systems by combining nano-silica (SiO2) and nitrification inhibitors. Background Technology

[0002] Paclobutrazol is a systemic fungicide and plant growth regulator affecting a wide range of plant species. Carbendazim is a benzimidazole fungicide and a systemic fungicide, widely used to control fungal diseases in intensive agriculture. However, carbendazim degrades slowly and has low water solubility, meaning its primary source in crops is soil. Paclobutrazol not only persists in soil for a long time but also has high residual levels, and its application may lead to its introduction into the food chain and harm human health. Studies have shown that residues of various agrochemicals (paclobutrazol, tebuconazole, carbendazim, and carbofuran) have been detected in crop samples, with carbendazim concentrations exceeding the set maximum residue levels. Due to its toxicity and environmental pollution effects, the European Union completely banned the production and use of carbendazim in November 2019. However, carbendazim has become one of the largest industrially produced fungicides in my country. Currently, my country's annual consumption of carbendazim exceeds 12,000 tons. Carbendazim is chemically stable, with a half-life in the soil environment typically ranging from 6 to 12 months. In greenhouse agriculture management, the recommended application frequency and dosage of carbendazim are once every 7 days and 5 mg / kg each time. -1 The recommended application frequency for paclobutrazol is multiple applications throughout the growing season, with the dosage depending on the plant species. However, improper use of paclobutrazol and carbendazim can lead to excessively high residues of these compound fungicides in soil and plants, causing serious toxicity to mammals. For example, compound fungicides (paclobutrazol and carbendazim) may persist in the environment as organic pollutants, altering microorganisms and metabolites in the soil-plant system. Furthermore, residues of paclobutrazol and carbendazim are widely detected in various foods, exceeding safety limits and posing a significant risk to human health. In addition, paclobutrazol residues can irritate the respiratory system, and prolonged exposure may lead to severe liver and kidney damage. Carbendazim residues may be toxic to hepatocytes and the thyroid gland, affect the reproductive system, and cause neurological disorders. Long-term exposure to compound fungicides can irritate the skin and mucous membranes, leading to various diseases.

[0003] The degradation of organic pollutants may be influenced by the nitrogen cycle. Applying nitrogen fertilizer can regulate the nitrogen cycle process and reduce organic pollutant residues. The application of nitrification inhibitors is primarily used as an effective measure to regulate the nitrogen cycle process and reduce nitrogen nutrient loss by inhibiting the conversion of ammonium nitrogen to nitrate nitrogen. The application of nitrification inhibitors can also promote the dissipation of organic pollutants by altering the composition and diversity of soil microbial communities. Furthermore, novel nanomaterials have shown effectiveness in reducing soil organic pollutant residues. Paclobutrazol and carbendazim are organic pollutants with different chemical structures and half-lives. Paclobutrazol is a weakly acidic substance and degrades easily under alkaline conditions, while carbendazim is a weakly basic compound and degrades easily under acidic conditions. In addition, the degradation process of complex pollutants is mainly dominated by microorganisms with different functions. Pseudomonas is the main microorganism affecting the degradation rate of paclobutrazol. The degradation of carbendazim is mainly dominated by Firmicutes and Penicillium. In addition, the application of silicon can stimulate the abundance of Pseudomonas, but inhibit the relative abundance of Firmicutes.

[0004] Current research largely focuses on the effects of individual application of nanomaterials and nitrification inhibitors on crop yield, with limited consideration of multiple factors including fungicide degradation, soil and endophytic microbial communities, non-targeted metabolomics (soil and plants), crop quality, and compound fungicide residues. Few studies have investigated the combined application of nano-SiO2 and nitrification inhibitors on reducing compound fungicide residues in soil-plant systems. Therefore, research is needed to explore the effects of nano-SiO2 alone or in combination with nitrification inhibitors on compound fungicide residues in soil-plant systems. Simultaneously, a systematic analysis of data on plant yield, quality indicators, compound fungicide residues in soil and plants, microorganisms, and metabolomics is required to ensure both plant yield and reduced mixed fungicide residues in soil and plants. Such results would deepen our understanding of the efficacy of nano-SiO2 and nitrification inhibitors, providing technical support for reducing the ecological risks of compound organic pollutant residues in soil-plant systems. Summary of the Invention

[0005] To promote plant growth and reduce the residues of compound fungicides paclobutrazol and carbendazim in soil and plants, this invention provides a method for reducing the residues of compound fungicides in soil and plants by combining nanomaterials and nitrification inhibitors.

[0006] The technical solution provided by this invention is as follows:

[0007] A composition that promotes plant growth and reduces the residual amount of compound fungicide in soil and plants, the composition comprising nanomaterials and nitrification inhibitors; the nanomaterials are nano-silica; the nitrification inhibitors are dicyandiamide; and the plant fungicides are paclobutrazol and / or carbendazim.

[0008] This invention also provides a method for reducing the residues of compound fungicides in soil and plants by combining nanomaterials and nitrification inhibitors, wherein the soil is treated with a composition containing nanomaterials and nitrification inhibitors; wherein the nanomaterials are nano-silica; the nitrification inhibitors are dicyandiamide; and the plant fungicides are paclobutrazol and carbendazim.

[0009] Preferably, the method further includes adding urea to the soil at an application rate of 100-300 mg N / kg. -1 Dry soil.

[0010] Preferably, the amount of nanomaterial applied relative to the soil is 0.1~0.5%w / w.

[0011] Preferably, the dosage of the nitration inhibitor is 1-4 mg N kg. -1 Dry soil.

[0012] Preferably, the soil moisture content is maintained at 50-70% WHC.

[0013] Preferably, the nitration inhibitor is dicyandiamide.

[0014] Preferably, the ratio of urea to test soil is 1:200.

[0015] Preferably, the nanomaterial is nano-SiO2.

[0016] Preferably, the ratio of nano-SiO2 to the test soil is 3:1000.

[0017] Preferably, the ratio of dicyandiamide to urea is 1:100.

[0018] The present invention also provides the use of the above-described composition or method in promoting plant growth.

[0019] The present invention also provides the application of the above-described composition or method in improving microbial diversity.

[0020] The present invention also provides the application of the above-described composition or method in improving the β-diversity of soil endophytic fungi.

[0021] The present invention also provides the application of the above-described composition or method in increasing the proline content of Chinese cabbage and the activity of soil β-glucosidase.

[0022] The application of a composition that promotes plant growth and reduces the residue levels of the compound fungicides paclobutrazol and carbendazim in soil-plant systems in promoting plant growth and influencing the residue levels of compound fungicides in soil and plants.

[0023] A planting method that promotes plant growth and reduces the residual levels of the compound fungicides paclobutrazol and carbendazim in a soil-plant system, wherein plants are planted in soil treated with a composition comprising nano-SiO2 and a nitrification inhibitor; the nitrification inhibitor comprising dicyandiamide.

[0024] Beneficial effects

[0025] (1) Taking the remediation of fungicide (paclobutrazol and carbendazim) pollution in agricultural soil as a starting point, this study investigates how to promote the degradation of fungicides in agricultural soil from multiple angles, in a comprehensive and three-dimensional manner through the combined use of biostimulation, nanomaterials, and nitrogen cycle regulation. The application of silicon materials may affect the growth and abundance of microorganisms, thereby regulating the degradation of pesticides in the soil. Nitrification inhibitors (DCDs) can regulate the soil nitrogen transformation process, thereby promoting crop growth and affecting the residual amount of compound fungicides in soil and plants. The application of nano-SiO2 significantly reduced the residual amounts of paclobutrazol and carbendazim in soil by 15.4% and 18.8%, and significantly reduced the residual amounts of paclobutrazol and carbendazim in plants by 6.6% and 33.5%, and significantly increased the biomass of Chinese cabbage. The application of DCDs also significantly reduced the residual amounts of paclobutrazol and carbendazim in soil by 12.1% and 3.8%, and reduced NO3 in plants by 20.0%. - -N content. Compared to DCD treatment alone, the combined application of nano-SiO2 and DCD significantly increased the biomass of Chinese cabbage by 74.5%. Nano-SiO2 alone significantly increased the soluble sugar content of vegetables by 48.3%. This invention combines microbiology and molecular biology techniques with soil stoichiometry to study the mechanisms regulating the degradation and translocation of paclobutrazol and carbendazim in soil and plants, demonstrating the interdisciplinary nature and technological advancement of plant nutrition, microbiology, and environmental science.

[0026] (2) There are few studies on the process and mechanism by which the application of nano-SiO2 and nitrification inhibitors promotes the degradation of compound fungicides. In the remediation of organic pollution in agricultural soils, the microbial community and biostimulation are the core targets of in-situ bioremediation strategies, and the processes of nitrification and denitrification are also coupled with the degradation of organic pollutants. By combining technologies from different disciplines, we can explore the process and mechanism by which biological and nanomaterial stimulation and nitrogen cycling promote the degradation of paclobutrazol and carbendazim at a higher and more complex overall level, so as to provide technical support for ensuring plant yield and quality, mitigating the environmental health risks of compound fungicides, and improving nutrient utilization.

[0027] (3) The present invention found that the application of nano-SiO2 and nitrification inhibitors can not only increase vegetable yield, but also reduce the residue of compound fungicides (paclobutrazol and carbendazim) in the soil-plant system. Therefore, nanomaterials and nitrification inhibitors have broad application prospects in agricultural systems. Attached Figure Description

[0028] Figure 1 This is the technical solution route of the present invention;

[0029] Figure 2 The effects of nano-silica and dicyandiamide on fungicide residues in soil were investigated. Among them, (a) was paclobutrazol; (b) was carbendazim residue and plant residues; (c) was paclobutrazol; (d) was carbendazim; CK was blank control; SiA was nano-silica application; DA was DCD application; SiDA was nano-silica + DCD.

[0030] Figure 3 The effects of nano-silica and dicyandiamide on plants are shown; (a) represents the yield of Chinese cabbage, (b) represents the soluble sugar content, (c) represents the proline content, and (d) represents the plant NO3 content. - -N content, (e) soil β-glucosidase activity; (f) soil electron transport system activity; CK, blank control; SiA, nano silica application; DA, DCD application; SiDA, nano silica + DCD;

[0031] Figure 4 (a) represents the taxonomic distribution of soil and microorganisms at the dominant phylum level (relative abundance > 0.1%); where (a) represents endophytic bacteria; (b) represents endophytic fungi; (c) represents bacteria in the soil-plant system; (d) represents fungal community structure; (e) represents soil and endophytic bacterial communities based on PCoA; and (f) represents β-diversity of soil and endophytic fungal communities.

[0032] Figure 5 The diversity of soil and endophytic bacteria and fungi; where (a) is the diversity index of soil and endophytic bacteria and fungi, (b) is the α-diversity of soil and endophytic bacteria, and (c) is the α-diversity of soil and endophytic fungi; CK, blank control; SiA, nano silica applied; DA, DCD applied; SiDA, nano silica + DCD;

[0033] Figure 6 The co-occurrence networks of soil microbial communities and soil metabolites are as follows: (a) is the co-occurrence network of soil bacterial communities and soil metabolites; (b) is the co-occurrence network of soil fungal communities and soil metabolites; (c) is the co-occurrence network of endophytic bacterial communities and plant metabolites; and (d) is the co-occurrence network of endophytic fungal communities and plant metabolites.

[0034] Figure 7 The results for different treatments; in (a) SiA, (b) DA and (c) SiDA treatments, the responses of soil metabolites, bacteria and fungal communities to the CK treatment were increased (+Log2 change) and decreased (-Log2 change).

[0035] Figure 8 The results for different treatments; in (a) SiA, (b) DA and (c) SiDA treatments, the responses of plant metabolites, bacterial and fungal communities to CK treatments were increased (+Log2 change) and decreased (-Log2 change).

[0036] Figure 9 Pearson correlation analysis was used to reveal the relationships between (a) soil abiotic and biotic properties, soil bacterial and fungal communities and metabolites, and (b) plant quality, endophytic bacterial and fungal communities and plant metabolites.

[0037] Figure 10 The results of the path analysis are as follows: The path analysis revealed (a) the quantitative associations between soil abiotic and biotic characteristics, ecosystem multifunctionality, soil metabolites, α- and β-diversity and functional potential of soil bacteria and fungi, and paclobutrazol and carbendazim residues in soil; (b) the standardized total effects of each factor on soil paclobutrazol and carbendazim residues; (c) the relationship between plant paclobutrazol and carbendazim residues and biomass; and (d) the standardized total effects of each factor on plant paclobutrazol and carbendazim residues and biomass. Data above the arrows represent standardized direct effects. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0039] Example 1

[0040] 1. Test soil:

[0041] The experimental soil was taken from the Chang'an Education Base of Hunan Agricultural University (E113°13′54″, N28°08′38″). We collected the topsoil from 0 to 20 cm depth, air-dried it, mixed it thoroughly, ground it, and sieved it (< 2 mm).

[0042] 2. Chemical reagents:

[0043] Nano-sized SiO2 (d = 30 ± 5 nm) was produced by Shanghai Maclean Biotechnology Co., Ltd. (Shanghai, China) with a purity greater than 99.5%. Dicyandiamide (DCD) was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China), chemically pure with a purity greater than 98.0%. Paclobutrazol and carbendazim standards (purity greater than 99.0%) were purchased from Shanghai Maclean Biotechnology Co., Ltd. (Shanghai, China). Paclobutrazol and carbendazim: Commercial wettable powders with 15% and 50% active ingredients, respectively, were used as fungicides. Chinese cabbage seeds (Pakchoi cambodiana).

[0044] 3. Test instruments:

[0045] High-performance liquid chromatograph (HPLC) (Agilent Technologies, Germany), ultraviolet spectrophotometer, continuous flow analytical system (Seal Analytical, UK), low-speed and high-speed centrifuges, drying oven, oil bath, etc.

[0046] 4. The specific technical solution of the present invention is as follows: Figure 1 The following is stated:

[0047] 4.1 Experimental Design:

[0048] The experimental design included four treatments: (1) a blank control without nano-SiO2 or DCD (CK); (2) nano-SiO2 alone (SiA); (3) DCD alone (DA); and (4) nano-SiO2 + DCD (SiDA). Each treatment consisted of six replicates. The application rate of nano-SiO2 relative to dry soil was 0.3% w / w. The urea application rate was 200 mg N / kg. -1 For dry soil, the recommended DCD application rate is 2 mg N / kg. -1 Dry soil.

[0049] Air-dried soil (2.5 kg) was placed in each flowerpot (34 cm long, 18.5 cm wide, and 15 cm high), and the soil moisture was adjusted to 60% WHC. Fifteen germinated Chinese cabbage seeds were selected, placed in the flowerpots, and transferred to a greenhouse, marked as day 0. After 19 days of growth, the Chinese cabbage seedlings were thinned to four seedlings per pot. Urea was dissolved in double-distilled water (ddH2O) and added to each flowerpot. Nano-SiO2 and DCD were dissolved in ddH2O and added to the corresponding treatments on day 20. Carbendazim was applied at 5 mg / kg every 7 days. -1 (On days 21, 28, 35, and 42), paclobutrazol was administered at a dose of 10 mg kg on days 28 and 35. -1Throughout the experiment, the experimental Chinese cabbage was monitored and watered with ddH2O. On day 47, samples of experimental soil and Chinese cabbage were collected and analyzed. The experimental soil in each pot was thoroughly mixed and separated into different subsamples for determination of: (1) abiotic and biotic characteristics of the soil; (2) paclobutrazol and carbendazim residues; (3) soil bacterial and fungal communities; and (4) non-targeted metabolomics.

[0050] 4.2 Determination of biomass, yield, and quality of Chinese cabbage

[0051] After the experimental crop matured, the planted crop was collected, washed, and drained. The fresh weight of the pak choi was measured and recorded as biomass. A portion was packaged and dried in an oven at 105 ℃ for 30 minutes to blanch, followed by drying at 70 ℃ to constant weight. The moisture content was calculated. Finally, the crop yield (yield based on both fresh and dry weight) was calculated. Additionally, the edible portion of the pak choi was weighed and its quality indicators (soluble sugar content, soluble protein, nitrate, total phenols, reduced glutathione, and proline) were determined.

[0052] 4.3 Determination of carbendazim content in soil and plant samples

[0053] Edible portions of the experimental plants were homogenized and, along with the experimental soil, freeze-dried. Acetonitrile was added for extraction, and the supernatant was rotary evaporated, diluted to volume with acetonitrile, and filtered through a 0.22 μm membrane. The concentration of carbendazim was then determined by HPLC-MS / MS. An external standard curve was plotted: the concentration of carbendazim was plotted on the x-axis, and the peak area on the y-axis. Separately, some soil samples were freeze-dried for the determination of carbendazim content in the soil. Experimental crops and soil (freeze-dried) were extracted with distilled water and methanol, centrifuged, and the supernatant was rotary evaporated, diluted to volume with methanol (chromatographically), filtered through a 0.22 μm organic filter, and the concentration of carbendazim was determined by HPLC-MS / MS.

[0054] 4.4 Determination of Paclobutrazol Content in Soil and Plants

[0055] Paclobutrazol residues in soil were extracted and measured. The procedure was as follows: the soil was freeze-dried and ground (< 0.18 mm). A soil sample (3.0 g), ddH2O (5.0 mL), and methanol (30.0 mL) were mixed, centrifuged, and filtered. After three extractions, the filtrate was collected and concentrated by rotary evaporation. Finally, the concentrated solution was brought to a final volume (10 mL), and the sample was filtered through an organic filter membrane (0.22 μm). The peak area of ​​paclobutrazol was detected using HPLC to calculate the paclobutrazol residue. Instrument parameters were as follows: sample injection volume: 20.0 μL; mobile phases A and B were acetonitrile solution and ddH2O, respectively; mobile phase flow rate: 0.5 mL / min.-1 Column temperature: 30 ℃, detection wavelength: 280 nm.

[0056] The residual amount of paclobutrazol in a sample of Chinese cabbage was analyzed. A 2.0 g sample of Chinese cabbage was ground, and 4 g of sodium chloride and 15 mL of acetonitrile were added, followed by vortexing. After centrifugation, 5 mL of the supernatant was collected and purified by adding 250 mg of ethylenediamine-N-propylsilane and 1 g of MgSO4 to a centrifuge tube. After vortexing, the solution was filtered through a 0.22 μm organic pinhole filter membrane and then subjected to HPLC-MS / MS. The mobile phases A and B were acetonitrile solution and ddH2O, respectively. The residual amount of paclobutrazol was calculated by quantifying the peak area of ​​paclobutrazol using HPLC-MS / MS.

[0057] 4.5 Microbial community sequencing and untargeted metabolomics analysis

[0058] Microbial community sequencing was performed according to methods described in relevant literature. DNA was extracted from the soil, and its concentration and quality were assessed. Qualified DNA samples were randomly fragmented into approximately 350 bp fragments using ultrasound. The entire library was prepared through end repair, sequencing adapter preparation, purification, and PCR amplification. After the library passed quality checks, high-throughput sequencing was performed. Plant samples were surface-sterilized, and DNA was extracted and its concentration and quality were assessed. Nested PCR was used to amplify the V5-V7 region of the 16 rRNA gene of endophytic bacteria: the first round of PCR used primers 779F and 1392R, producing a fragment of approximately 593 bp; the second round used primers 779F and 1193R, producing a fragment of approximately 394 bp. Primers ITS1F and ITS2R were used to amplify the ITS fragment, the internal transcriptional spacer region of plant endophytic fungi. The PCR products were electrophoresed, gel-cleaved, and used to construct sequencing libraries before high-throughput sequencing. The sequencing data was filtered to obtain high-quality data.

[0059] Soil or plant samples were mixed with grinding beads in 2 mL centrifuge tubes, and metabolites were extracted using an extraction solution containing four internal standards. The solution was filtered through a 0.22 μm polycarbonate membrane, and the supernatant from both soil and plant samples was carefully transferred to vials for metabolite analysis. All metabolomics data were analyzed using the Majorbio I-Sanger cloud platform. The magnitude variation (Δmetabolite abundance) was quantified by scaling metabolite peak height from 0 to 1, after which positive metabolites were selected from soil and plant samples. The topological features and visualization of the bacterial, fungal, and metabolite co-occurrence network in soil and plant samples were estimated and performed using the igraph software package and Gephi.

[0060] 4.6 Data Analysis Methods

[0061] Statistical methods: One-way ANOVA with minimum significance test was used to compare variables among different treatments. Correlation analysis was used to reveal the comprehensive relationships between soil biotic and abiotic properties, paclobutrazol and carbendazim content, microbial activity and microbial community structure, and soil and plant metabolomes. Path analysis was used to analyze the direct and indirect relationships between paclobutrazol and carbendazim residues, different forms of nitrogen, functional gene abundance, soil and endophytic microorganisms, and metabolomes.

[0062] Results analysis:

[0063] 1. Figure 1 This is a roadmap of the technical solution of the present invention.

[0064] 2. Residue levels of paclobutrazol and carbendazim in soil and plant samples

[0065] Compared with the control (CK) treatment, the soil residues of paclobutrazol and carbendazim in the SiA treatment were significantly reduced by 15.4% and 18.8%, respectively. Figure 2 (a-2b) The residues of paclobutrazol and carbendazim in plants treated with SiA decreased significantly by 6.6% and 33.5%, respectively. Figure 2 c-2d). The soil paclobutrazol and carbendazim residues in the DA treatment were significantly lower than those in the CK treatment (12.1% and 3.8% lower, respectively). Compared with CK (80 ± 3 mg kg... -1 Compared to dry matter, DA treatment significantly reduced carbendazim residue in plants by 35.7% ( Figure 2 a-2b). The paclobutrazol residue in plants treated with SiDA was significantly lower than that treated with DA (161 ± 21 mg kg). -1 Dry matter (reduced by 16.4%), but the residue of carbendazim in the SiDA treatment was significantly higher than that in the DA treatment ( Figure 2 c) indicates that nano-SiO2 selectively enhances the performance of DCD in reducing plant complex organic pollutants, thereby reducing paclobutrazol residues, but hindering the degradation of carbendazim in plants.

[0066] 3. Yield, quality, and soil enzyme activity of Chinese cabbage

[0067] With DA (302 ± 134 kg hm) -2 Compared to dry matter, SiDA treatment significantly increased the yield of Chinese cabbage by 74.5% ( Figure 3 a). Compared with CK, the soluble sugar content of SiA treatment increased significantly by 48.3% ( Figure 3 b). The proline content of Chinese cabbage treated with SiDA was higher than that treated with SiA ( Figure 3 c). Compared to CK, DA treatment of bok choy NO3 --N content decreased significantly by 20.0%, but SiDA-treated bok choy NO3 content was higher. - -N content was significantly higher than that in DA treatment ( Figure 3 d). The soil β-glucosidase activity in the DA treatment was 93.5% of that in the CK treatment. Compared with the DA treatment, the soil β-glucosidase activity of the SiDA counterpart was significantly increased by 18.9%. Figure 3 e). Significant differences in soil electron transport system activity among the four treatments ( Figure 3 f).

[0068] 4. Soil and endophytic bacteria and fungi and their functional genes

[0069] The relative abundance of acidobacteriota in soil treated with SiDA was 1.77 times that of soil treated with CK. Figure 4 a). The relative abundance of Proteobacteria in the soil treated with DA was significantly higher than that in the CK treatment (0.35 ± 0.06) (21.5% higher). Compared with the DA treatment, the relative abundance of endophytic Acidobacteriota in the SiDA treatment increased significantly by 75.0%. The relative abundance of endophytic fungus Ascomycota in the DA treatment was 91.3% higher than that in the CK treatment (0.24 ± 0.15). Figure 4 b). DA treatment significantly altered the community structure of soil and endophytic bacteria. Significant differences in bacterial and fungal community structure were observed in all 48 samples (24 soil and 24 plant samples). Figure 4 c and 4d). Endophytic bacterial β-diversity was significantly lower in SiDA-treated patients than in DA-treated patients (c and 4d). Figure 4 e); the β-diversity of endophytic fungi in SiDA treatment was significantly higher than that in DA treatment ( Figure 4 f).

[0070] The ACE abundance index of endophytic fungi in the DA treatment was significantly higher than that in the CK treatment (136 ± 53) (35.8% higher). Figure 5 a). Soil and endophytic bacterial community α-diversity showed no significant differences across the four treatments ( Figure 5 b). The α-diversity of endophytic fungal communities in the SiA treatment was significantly higher than that in the CK treatment ( Figure 5 c).

[0071] 5. Co-occurrence networks of soil and plant metabolomes and microbial communities

[0072] Four co-occurrence networks were constructed based on soil and endophytic bacteria or fungi and their metabolites (40 from soil and 40 from plants). Figure 6a-6d). The dichotomous co-occurrence network of soil fungi and soil metabolites, as well as the soil fungal OTUs, are connected to the top 40 soil metabolites through 976 nodes and 3668 edges, with an average of 4 edges per node. These networks include 8 soil fungal phyla and 36 soil metabolites ( Figure 6 b). A bipartite co-occurrence network of endophytic fungi and plant metabolites, where endophytic fungal OTUs are connected to the top 40 plant metabolites through 353 nodes and 403 edges, including 2 endophytic fungal phyla and 31 plant metabolites ( Figure 6 d). The number of co-occurrence network nodes of endophytic fungi and plant metabolites in the DA treatment was significantly higher than that in the CK treatment (17.5% higher). Therefore, the addition of the nitrification inhibitor DCD significantly increased the number of co-occurrence network nodes of endophytic fungi and plant metabolites. Our study shows that applying the nitrification inhibitor DCD can make the microbial community more stable.

[0073] 6. Chemical changes in microorganisms and metabolites caused by the treatment.

[0074] The taxonomic characteristics of microorganisms and metabolites responding to different treatments depend on whether they are positively reacting (increased abundance) or negatively reacting (decreased abundance). In the SiA treatment, positive reactants included Nitrospirota (0.31), Chytridiomycota (0.31), and heliangin (0.06) in soil samples, while negative reactants included Gemmatimonadota (-0.48), Rozelomycota (-1.74), and erucamide (-0.04), respectively. Figure 7 a). In DA treatment, the positive reactants were Proteobacteria, Glomeromycota, and crocin 5, while the negative reactants in the soil were Acidobacteriota, Chytridiomycota, and dodecyl sulfate (a). Figure 7 b). In SiDA treatment, the positive and negative reactions were Bdellovibrionota (0.13), Kickxellomycota (2.36), Raffinose (0.04), Gemmatimonadota (-0.56), Glomeromycota (-0.64), and ononin (-0.07), respectively. Figure 7 c).

[0075] The positive responses to SiA treatment in plant samples were Verrucomicrobiota (7.59), Ascomycota (0.58), and cholic acid (0.05), while the negative responses were Bacteroidota (-1.59), Chytridiomycota (-2.08), and Hypoxanthine (-0.11). Figure 8 a). The positive and negative responses to DA treatment in plant samples were Gemmatimonadota (+), Ascomycota (+), montanol (+), Acidobacteriota (-), Chytridiomycota (-) and citric acid (-), respectively. Figure 8 b). The positive and negative responses to SiDA treatment were for verrucous microbes (7.65), bile acids (0.06), Bacteroides (-1.27), Chlamydia (-7.33), and hypoxanthine (-0.11). Figure 8 c).

[0076] 7. Overall Relationship of All Indicators

[0077] The relative abundance of Proteobacteria and Firmicutes in soil, as well as the edges of the co-occurrence network of soil metabolites, bacteria, or fungi, were negatively correlated with soil paclobutrazol and carbendazim residues. Figure 9 a) Soil β-glucosidase activity and Basidiomycota phylum were negatively correlated with soil paclobutrazol residues. Plant proline content, relative abundance of endophytic bacteria Proteobacteria and Acidobacteriota, estimated abundance values ​​of ACE and Chao1, as well as fungal Basidiomycota and plant metabolites hexadecylphosphonic acid and L-tyrosine were negatively correlated with plant paclobutrazol residues, but positively correlated with plant carbendazim residues. Figure 9 b). The relative abundance of endophytic myxococcota and cyclopentanol and hexadecylphosphine were significantly positively correlated with plant biomass. Figure 9 ).

[0078] Path analysis (χ) 2 = 4.003, df = 2, P = 0.135) indicates that increased ecosystem multifunctionality and soil bacterial α-diversity can reduce soil paclobutrazol residues, while promoted soil fungal β-diversity can reduce soil carbendazim residues. Figure 10a). Soil biotic and abiotic properties and soil bacterial functional potential have a positive standardized effect on soil carbendazim and paclobutrazol residues ( Figure 10 b). Path analysis (χ²) 2 = 3.572, df = 3, P = 0.312) indicates that increased soil physicochemical properties and ecosystem multifunctionality can significantly promote the biomass of Chinese cabbage, and ecosystem multifunctionality and paclobutrazol residues in plants have a significant negative correlation (10c). Endophytic fungal α-diversity has a negative standardized effect value on paclobutrazol and paclobutrazol residues and biomass in plants ( Figure 10 d).

[0079] This invention focuses on the remediation of fungicide (paclobutrazol and carbendazim) pollution in agricultural soils. It investigates a multi-faceted, comprehensive, and three-dimensional approach to promoting the degradation of compound fungicides in agricultural soils through a combination of nitrogen cycle regulation, biostimulation, and nanomaterial stimulation. Nitrification inhibitors (DCDs) can regulate soil nitrogen transformation processes, thereby promoting crop growth and influencing fungicide residues in soil and plants. This invention combines microbiology and molecular biology techniques with soil stoichiometry to study the mechanisms regulating the degradation and translocation of paclobutrazol and carbendazim in soil and plants, demonstrating interdisciplinary collaboration and technological advancement.

[0080] This invention addresses the limited research on the process and mechanism by which the application of nano-SiO2 and nitrification inhibitors promotes the degradation of compound fungicides. In the remediation of organic pollution in agricultural soils, microbial communities, metabolomics, and biostimulation are core targets of in-situ bioremediation strategies, and the processes of nitrification and denitrification are also coupled with the degradation of organic pollutants. This invention utilizes a combination of techniques from different disciplines to explore the process and mechanism by which nanomaterials, biostimulation, and nitrogen cycling promote the degradation of compound fungicides (paclobutrazol and carbendazim) at a higher and more complex overall level. This provides technical support for ensuring the yield and quality of agricultural products, mitigating the environmental health risks of fungicides, and improving nutrient utilization.

[0081] This invention discovers that the application of nano-SiO2 and nitrification inhibitors can not only increase the yield of Chinese cabbage, but also reduce the residues of compound fungicides (paclobutrazol and carbendazim) in the soil-crop system. Therefore, nanomaterials and nitrification inhibitors have broad application prospects in agricultural systems.

Claims

1. A composition that promotes plant growth and reduces the residual amount of compound fungicide in soil and plants, characterized in that, The composition comprises nanomaterials and nitrification inhibitors; the nanomaterials are nano-silica; the nitrification inhibitors are dicyandiamide; and the plant fungicide is paclobutrazol and / or carbendazim.

2. A method for reducing soil-plant residues of compound fungicides by combining nanomaterials and nitrification inhibitors, characterized in that, Soil is treated with a composition containing nanomaterials and nitrification inhibitors; the nanomaterials are nano-silica; the nitrification inhibitors are dicyandiamide; and the plant fungicides are paclobutrazol and carbendazim.

3. The method for reducing soil-plant residues of compound fungicides by combining nanomaterials and nitrification inhibitors according to claim 2, characterized in that, The method also includes adding urea to the soil at an application rate of 100-300 mg / kg. -1 Dry soil.

4. The method for reducing soil-plant residues of compound fungicides by combining nanomaterials and nitrification inhibitors according to claim 2, characterized in that, The nanomaterial is applied at a rate of 0.1-0.5% w / w relative to the soil.

5. The method for reducing soil-plant residues of compound fungicides by combining nanomaterials and nitrification inhibitors according to claim 2, characterized in that, The dosage of the nitration inhibitor is 1-4 mg N kg. -1 Dry soil.

6. The method for reducing soil-plant residues of compound fungicides by combining nanomaterials and nitrification inhibitors according to claim 2, characterized in that, Maintain soil moisture at 50-70% WHC.

7. The use of the composition of claim 1 or the method of any one of claims 2 to 6 in promoting plant growth.

8. The use of the composition of claim 1 or the method of any one of claims 2 to 6 in improving microbial diversity.

9. The use of the composition of claim 1 or the method of any one of claims 2 to 6 in improving the β-diversity of soil endophytic fungi.

10. The use of the composition of claim 1 or the method of any one of claims 2 to 6 in increasing the proline content of Chinese cabbage and the activity of soil β-glucosidase.

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