Application of difenoconazole as a fungal denitrification inhibitor in controlling soil nitrous oxide release

Abstract

The present invention discloses the use of difenoconazole as a fungal denitrification inhibitor to control nitrous oxide release in soil, relating to the field of pharmaceutical application technology. In the present invention, difenoconazole targets and regulates the fungal denitrification process, effectively reducing the emission of the greenhouse gas N₂O (nitrous oxide) after nitrogen fertilizer application in farmland (i.e., in flooded farmland soil environments). Specifically, under laboratory conditions, difenoconazole reduced soil N₂O emissions by 75-93%; under experimental conditions in rice fields, difenoconazole reduced N₂O emissions by 54%.

Description

Technical Field

[0001] The invention relates to the technical field of drug application, and in particular to the application of difenoconazole as a fungal denitrification inhibitor in controlling the release of nitrous oxide in soil. Background Art

[0002] Nitrous oxide (N2O) is a greenhouse gas with a strong radiative effect, contributing up to 6.2% to global temperature rise. Therefore, to mitigate the greenhouse effect, regulating and reducing N2O emissions is urgent.

[0003] Human agricultural activities have exacerbated N2O emissions from farmland soils. Microbial denitrification in farmland soils is a significant source of N2O emissions, with fungal denitrification contributing approximately 20-25% of total N2O emissions. The production of N2O from microbial denitrification stems from the lack of N2O reductase (NOS) within fungal cells. Therefore, fungal denitrification typically produces N2O as the final product. Consequently, microbial denitrification in farmland soils significantly increases greenhouse gas emissions from farmland. Studies have shown that flooded farmland soils have higher N2O emission rates from fungal denitrification. Previous research indicates that fungal denitrification accounts for 51-63% of total N2O emissions from rice fields nationwide. Therefore, reducing N2O release from fungal denitrification is crucial for reducing greenhouse gas emissions from farmland and mitigating global climate change.

[0004] Currently, the means of controlling N2O release from farmland soil lies in regulating nitrification. The main way to regulate nitrification is to apply nitrification inhibitors DMPP (3,4-dimethylpyrazole phosphate), DCD (dicyandiamide) and Nitrapyrin (2-chloro-6-(trichloromethyl)pyridine). However, existing nitrification inhibitors can only regulate nitrification, but are difficult to inhibit the denitrification process that actually produces more N2O. In the flooded farmland soil environment, the N2O emissions produced by denitrification are still very high. Therefore, in order to further control the release of N2O in agricultural activities, it is necessary to study the inhibitors of the denitrification process. Summary of the Invention

[0005] The present invention discloses the use of difenoconazole as a fungal denitrification inhibitor in controlling the release of nitrous oxide in soil. Difenoconazole targets and regulates the fungal denitrification process to reduce the problem of increased greenhouse gas N2O emissions after nitrogen fertilizer application in farmland (i.e., in flooded farmland soil environments).

[0006] The technical solution adopted in the present invention is as follows:

[0007] Application of difenoconazole as a fungal denitrification inhibitor in controlling nitrous oxide release from soil.

[0008] Furthermore, the soil is flooded farmland soil.

[0009] Furthermore, the soil is paddy field soil.

[0010] A soil fungal denitrification inhibitor comprising difenoconazole.

[0011] In summary, compared with the prior art, the present invention has the following advantages and beneficial effects:

[0012] 1. The present invention discloses the use of difenoconazole as a fungal denitrification inhibitor in controlling the release of nitrous oxide in soil: difenoconazole can target and regulate the fungal denitrification process, thereby effectively reducing the emission of the greenhouse gas N2O after nitrogen fertilizer application in farmland (i.e., in flooded farmland soil environment). Specifically, under laboratory conditions, difenoconazole can reduce soil N2O emissions by 75-93%; under experimental conditions in rice fields, difenoconazole reduced N2O emissions in rice fields by 54%;

[0013] 2. The present invention discloses a soil fungal denitrification inhibitor in farmland soil, which effectively inhibits the denitrification process mediated by microorganisms in farmland soil;

[0014] 3. Compared with traditional nitrification inhibitors, the present invention provides a new idea of ​​using denitrification inhibitors to reduce N2O emissions. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 : This is a bar graph of the N2O release of each treatment group in the laboratory soil incubation experiment in Example 1, wherein a is the control group and the difenoconazole alone treatment group, b is the control group of the bacterial denitrification treatment group and the difenoconazole + bacterial denitrification treatment group, c is the control group of the fungal denitrification treatment group and the difenoconazole + fungal denitrification treatment group, and d is the control group of the abiotic denitrification treatment group and the difenoconazole + abiotic denitrification treatment group;

[0016] Figure 2 This is the layout diagram of the experimental device for monitoring N2O emissions from farmland using the static dark box method in Example 2, where a is a physical diagram of the box and b is a diagram of the box placed in rice fields;

[0017] Figure 3 Graph showing the N2O release of the difenoconazole group and the blank group in the rice field cultivation experiment in Example 2, wherein a is a line graph showing the N2O release at different times after application of urea and difenoconazole, and b is a statistical bar graph showing the N2O release. DETAILED DESCRIPTION

[0018] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with various embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. That is, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments.

[0019] The term "Example" is used herein specifically to describe any embodiment as "exemplary" and should not be construed as superior or preferable to any other embodiment. Performance indicators in the embodiments of this invention were tested using conventional test methods in the art unless otherwise specified. The terms used in this invention are intended solely to describe specific embodiments and are not intended to limit the scope of this disclosure.

[0020] Unless otherwise specified, the technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which the invention belongs; other raw materials, reagents, experimental methods and technical means not otherwise specified in the present invention refer to the raw materials and reagents commonly used by those skilled in the art, as well as the commonly used experimental methods and technical means.

[0021] Example 1

[0022] This example provides a laboratory soil culture experiment on the regulation of fungal denitrification by difenoconazole:

[0023] 1. Set up blank group, bacterial denitrification treatment group, fungal denitrification treatment group and abiotic denitrification treatment group

[0024] (1) Blank control group

[0025] 1 g (dry weight) of soil sample was placed in a 12 mL Labco test tube, and 1 mL of a solution containing glucose (carbon source) and sodium nitrate (nitrogen source) was added. The specific concentrations were glucose 180 mg / kg (soil) and sodium nitrate 100 mg / kg (soil).

[0026] (2) Bacterial denitrification treatment group

[0027] 1 g (dry weight) of soil sample was placed in a 12 mL Labco test tube and 1 mL of a solution containing glucose (carbon source), sodium nitrate (nitrogen source), and cycloheximide (fungal inhibitor) was added. The specific concentrations were 180 mg / kg (soil) of glucose, 100 mg / kg (soil) of sodium nitrate, and 10 mg / g (soil) of cycloheximide.

[0028] (3) Fungal denitrification treatment group

[0029] 1 g (dry weight) of soil sample was placed in a 12 mL Labco test tube and 1 mL of a solution containing glucose (carbon source), sodium nitrate (nitrogen source), and streptomycin (bacterial inhibitor) was added. The specific concentrations were glucose 180 mg / kg (soil), sodium nitrate 100 mg / kg (soil), and streptomycin 6 mg / g (soil).

[0030] (4) Abiotic denitrification treatment group

[0031] 1g (dry weight) of soil sample was placed in a 12mL Labco test tube and 1mL of a solution containing glucose (carbon source), sodium nitrate (nitrogen source), cycloheximide (fungal inhibitor), and streptomycin (bacterial inhibitor) was added. The specific concentrations were 180mg / kg (soil) of glucose, 100mg / kg (soil) of sodium nitrate, 10mg / g (soil) of cycloheximide, and 6mg / g (soil) of streptomycin.

[0032] (5) Difenoconazole alone treatment group

[0033] 1 g (dry weight) of soil sample was placed in a 12 mL Labco test tube and 1 mL of a solution containing glucose (carbon source), sodium nitrate (nitrogen source), and difenoconazole was added. The specific concentrations were 180 mg / kg (soil) of glucose, 100 mg / kg (soil) of sodium nitrate, and 1 mg / kg (soil) of difenoconazole.

[0034] (6) Difenoconazole + bacterial denitrification treatment group

[0035] 1g (dry weight) of soil sample was placed in a 12mL Labco test tube and 1mL of a solution containing glucose (carbon source), sodium nitrate (nitrogen source), cycloheximide (fungal inhibitor), and difenoconazole was added. The specific concentrations were 180mg / kg (soil) of glucose, 100mg / kg (soil) of sodium nitrate, 10mg / g (soil) of cycloheximide, and 1mg / kg (soil) of difenoconazole.

[0036] (7) Difenoconazole + fungal denitrification treatment group

[0037] 1g (dry weight) of soil sample was placed in a 12mL Labco test tube and 1mL of a solution containing glucose (carbon source), sodium nitrate (nitrogen source), streptomycin (bacterial inhibitor), and difenoconazole was added. The specific concentrations were 180mg / kg (soil) of glucose, 100mg / kg (soil) of sodium nitrate, 6mg / g (soil) of streptomycin, and 1mg / kg (soil) of difenoconazole.

[0038] (8) Difenoconazole + abiotic denitrification treatment group

[0039] 1g (dry weight) of soil sample was placed in a 12mL Labco test tube and 1mL of a solution containing glucose (carbon source), sodium nitrate (nitrogen source), cycloheximide (fungal inhibitor), streptomycin (bacterial inhibitor), and difenoconazole was added. The specific concentrations were glucose 180mg / kg (soil), sodium nitrate 100mg / kg (soil), cycloheximide 10mg / g (soil), streptomycin 6mg / g (soil), and difenoconazole 1mg / kg (soil).

[0040] 2. Determine the N2O release of each treatment group

[0041] The Labco test tube was flushed with nitrogen until it filled the entire headspace. The tube was then placed in an anaerobic glove box and incubated for 72 hours, with the moisture content controlled at 70% at 25 ± 0.5°C. The N2O concentration in the vial was measured using a gas chromatograph with an electron capture detector (ECD) using high-purity He as the carrier gas at a flow rate of 40 mL / min, a column temperature of 60°C, an inlet temperature of 120°C, and a detector temperature of 300°C.

[0042] The results are as follows Figure 1 As shown in the results, under laboratory conditions, difenoconazole can significantly reduce the total N2O release in the soil by 76.9%, of which the inhibition rate of fungal denitrification release is as high as 93.3%. However, it has no significant effect on the N2O release in the soil of the bacterial denitrification treatment group and the abiotic denitrification treatment group, indicating that difenoconazole can target and regulate the fungal denitrification process, thereby reducing the release of N2O in the soil.

[0043] Example 2

[0044] This example provides a rice field cultivation experiment on the regulation of fungal denitrification by difenoconazole:

[0045] In Ansheng Town, Liangping District, Chongqing City (30°41'46"N, 107°44'25"E), at an altitude of 453 meters, two rice fields (rice variety: Weiliangyouyuzhan) with equivalent soil conditions (total organic matter content 17.47±2.90g / kg, total nitrogen content 1.17±0.02g / kg, total phosphorus content 0.72±0.02g / kg, pH value 6.83±0.10) were selected and named as site A (difenoconazole group) and site B (blank group).

[0046] Urea (30 kg / mu) and difenoconazole (15 g / mu, 0.05% urea application rate) were applied to site A. Figure 2 As shown in the figure, the static dark box method is used to monitor the N2O release from farmland: the box is made of 6mm thick polyvinyl chloride material with a bottom area of ​​0.09m 2The volume is 27L. The outside of the box is wrapped with sponge and tin foil to prevent drastic temperature changes in the box due to sunlight during sampling. On the day of transplanting the rice seedlings, a square base was pre-buried in the center of each plot. The base was embedded 5cm deep in the soil as the sampling point. The upper end of the base consists of a groove approximately 3cm deep for placing the static box body. It is sealed during sampling to prevent the exchange of ambient air with the gas in the box. The N2O content was measured 0.5, 1, 2, 4, 8, 12, 24, 48, 72, 96, 120, 144, 168, 216, 164, 336, 408, and 504h after applying urea.

[0047] In site B, only urea (30 kg / mu) was applied, and the static dark box method was also used to monitor N2O emissions from farmland.

[0048] The results are as follows Figure 3 As shown in the results, under the conditions of rice fields, the difenoconazole group reduced the N2O emission in the rice fields by 54% compared with the blank group.

[0049] The above-described embodiments merely represent specific implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of protection of the present application. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the technical concept of the present application, and all such variations and improvements fall within the scope of protection of the present application.

Claims

1. A method for reducing the release of soil N2O, characterized in that: Streptomycin and difenoconazole were applied to the soil, wherein the amount of streptomycin was 6 mg per gram of soil, and the amount of difenoconazole was 1 mg per kilogram of soil.

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