Application of dihydromicanthone in the preparation of nitrification inhibitors

Abstract

The present invention relates to the application of dihydrochamolide in the preparation of plant-derived nitrification inhibitors, and belongs to the field of nitrification inhibitors. The present invention indicates that the compound has a good nitrification inhibition effect through bacterial pure culture and soil addition experiments, and can effectively inhibit the activity of ammonia oxidizing bacteria as a biological nitrification inhibitor, hinder the ammonia oxidation process, and is used. The nitrification inhibitor of the present invention is derived from plants, has high biological safety, stable chemical structure and significant nitrification inhibition effect, is not easy to lose in the soil, has stability and high efficiency, and can solve the problems of poor stability, easy degradation, toxicity and easy pollution of conventional chemically synthesized nitrification inhibitors. At the same time, the present invention also relates to at least one nitrification inhibitor agricultural composition including dihydrochamolide. After the agricultural composition is applied to the soil, it effectively inhibits the nitrification process of ammonium nitrogen, so that nitrogen conversion is effectively regulated.

Description

Technical field:

[0001] The invention relates to the technical field of nitrification inhibitors, and relates to the application of dihydrochamolide as a nitrification inhibitor. Background technology:

[0002] NH 4 + Nitrogen fertilizer in the form of NH 4 + In the soil, it is easily converted into NO by nitrifying microorganisms through nitrification. 3 - , NO 3 - It is not easily adsorbed by the soil, but enters the water body through leaching, causing environmental pollution and other problems. At the same time, NO 3 - In gaseous form, N 2 and N 2 O form loss, N 2 O is a greenhouse gas that plays an important role in global temperature rise. In my country's farmland ecosystem soil, the average nitrogen fertilizer utilization rate is 20%-40%, of which about 60% of nitrogen fertilizer loss comes from NH 4 + The leaching and gas loss of nitrification and denitrification processes can improve NH 4 + Improving the utilization rate of nitrogen fertilizer and reducing nitrogen loss are issues that need to be addressed urgently.

[0003] Since the 1970s, countries around the world have been working to improve NH 4 + various nitrogen fertilizer management measures have been developed and a series of measures have been carried out to delay or inhibit NH 4 + Nitrification research, in which nitrogen fertilizer combined with nitrification inhibitors can significantly inhibit soil nitrification, delay the nitrification process by inhibiting the activity of soil subsoil bacteria and other microorganisms, and hinder NH 4 + To NO 2 - and NO 3 - Conversion, thereby reducing NO 3 - Leaching losses and N 2 With N 2 O and other gaseous nitrogen losses. Maintaining high concentrations of NH 4 + The content lasts for a long time, which can effectively improve the utilization rate of ammonium nitrogen fertilizer.

[0004] At present, synthetic nitrification inhibitors mainly include sulfur-containing compounds, acetylene and acetylene substituents, cyanamide compounds and heterocyclic nitrogen compounds, among which the most common nitrification inhibitors are acetylene, 2-chloro-6-trichloromethylpyridine (CP) and dicyandiamide (DCD), 3,4-dimethylpyrazole phosphate (DMPP), etc. However, most of the above nitrification inhibitors have problems such as unstable performance, toxicity, and easy degradation. Therefore, it is difficult to promote their use in large-scale agricultural ecosystems, and long-term application may cause environmental pollution. Compared with artificial chemically synthesized nitrification inhibitors, biological nitrification inhibitors derived from plant secretions or extracts are natural and easily available compounds with little impact on the ecological environment. At the same time, they have the functions of promoting the mineralization of soil organic nitrogen and increasing NH 4 + Therefore, screening new high-efficiency and low-toxic nitrification inhibitors can contribute to the green development of agriculture. Summary of the invention:

[0005] The purpose of the present invention is to provide the use of dihydromicanthone in the preparation of nitrification inhibitors to solve the problems of the above-mentioned existing nitrification inhibitors. The substance inhibits the nitrification process of the soil by inhibiting the activity of nitrite bacteria, improves the utilization rate of ammonium nitrogen, and reduces the leaching of nitrate nitrogen and the emission of greenhouse gases produced by denitrification. The nitrification inhibitor of the present invention is a new natural fat-soluble substance with a stable chemical structure derived from plants, which provides a certain basis for the development of new high-efficiency and low-toxic nitrification inhibitors.

[0006] Technical solution:

[0007] Application of dihydromicanthone in the preparation of plant-derived nitrification inhibitors.

[0008] Furthermore, the dihydromicanthone is secreted from the roots of Mikania micrantha.

[0009] Furthermore, the dihydromicanthone (molecular formula: C 15 H 16 O 6 , molecular weight is 292.28), and the structural formula is shown in formula (1):

[0010]

[0011] Furthermore, the nitrosating microorganism inhibited by dihydromichanolide is Nitrosomonas europaea, ATCC 19718.

[0012] Furthermore, the use concentration of the dihydromicanthone is 20-140 μm; dihydromicanthone having a nitrogen content of 1%-10% by weight of N is added to the nitrogen-containing fertilizer.

[0013] The nitrogen-containing fertilizer is an ammonium nitrogen fertilizer such as ammonium chloride, ammonium sulfate, ammonium bicarbonate, liquid ammonia (ammonia water), monoammonium phosphate or diammonium phosphate, preferably ammonium sulfate.

[0014] The application method includes: directly adding dihydromicanthone to N fertilizer for use, or using dihydromicanthone in preparing fertilizer for nitrosating microorganisms inhibited by ammonium nitrogen fertilizer, wherein the nitrosating microorganisms are Nitrosomonas.

[0015] Furthermore, the application method comprises: adding dihydromichanolide to fertilizer to prepare a plant-derived inhibitor fertilizer.

[0016] Furthermore, the dihydromichanolide can effectively inhibit the nitrification process of ammonium nitrogen and promote the biological and non-biological fixation of ammonium nitrogen in the soil, thereby improving the nitrogen utilization rate of fertilizers and thereby increasing crop yield and dry matter weight.

[0017] Technical effects:

[0018] (1) The compounds of the present invention can be used as nitrification inhibitors, enriching the types of nitrification inhibitors in the field of plant sources, and can effectively inhibit the activity of nitrite-forming microorganisms at low doses, thereby reducing nitrogen loss and environmental problems caused by the nitrification process in farmland soil;

[0019] (2) The compound of the present invention is a fat-soluble compound with a stable chemical structure and is not easily lost in soil and water, thus ensuring stability and high efficiency in application.

[0020] (3) The nitrification inhibitor of the present invention is derived from plants and is a "green" biosafe nitrification inhibitor. It also has the function of promoting soil NH 4 + fixation, in order to improve nitrogen utilization and maintain a high NH 4 + The effect of content can solve the problems of toxicity and easy degradation of conventional chemically synthesized nitrification inhibitors, and provide new methods and ideas for biological ecological technology to regulate the nitrogen transformation process in farmland soil and improve nitrogen utilization efficiency. Description of the drawings:

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1The graph shows the inhibitory effect of different concentrations of dihydromichanolide and DCD on the activity of ammonia oxidizing bacteria Nitrosomonas europaea (ATCC 19718);

[0023] Figure 2 This is a graph showing the inhibitory effects of dihydromicanthone, dicyandiamide and micanthone on soil nitrification; Specific implementation method:

[0024] The following are various exemplary embodiments of the present invention. The detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features and embodiments of the present invention.

[0025] Example 1 Experiment on the inhibitory effect of different concentrations of dihydromichanolide on ammonia oxidizing bacteria Nitrosomonas europaea (ATCC19718):

[0026] (1) Dihydromicanthide was purchased from Shanghai Yuanye Biotechnology Co., Ltd. and was of superior purity; dicyandiamide was purchased from Aladdin Company and was of analytical purity of more than 98%.

[0027] (2) Microbial strain: Nitrosomonas europaea (ATCC 19718), purchased from Shenzhen Zike Biotechnology Co., Ltd.

[0028] (3) Preparation of test solution

[0029] Use 20 mM HEPES buffer to prepare dihydromichanolide solutions with concentrations of 5 μM, 10 μM, 20 μM, 40 μM, 60 μM, 80 μM, 100 μM, 140 μM, and 180 μM, and store in a dark place and refrigerate until use.

[0030] (4) Preparation of resuspended bacterial solution: In HEPES pure medium (1L liquid medium contains 2.5g (NH4) 2 SO 4 ,0.5gKH 2 PO 4 , 5 mg CaCl 2 ·2H 2 O, 0.1 g MgSO 4 .7H 2 O, 0.5 g NaHCO 3 European Nitrosomonas was added into a medium (1 g NaCl, 75 mg Fe-EDTA, 11.92 g HEPES), and the culture was carried out at 200 rpm under light-free conditions. The culture solution was collected for 7 days and centrifuged to obtain a concentrated bacterial solution.

[0031] (5) Nitrite inhibition experiment

[0032] Take 50 μL of the above-mentioned dihydromichanolide solution with different concentrations, add 950 μL of sterile water and 1 mL of resuspended bacterial solution respectively, and culture at 25°C in the dark for 2 hours. Then, add 250 μL of the mixed solution after the reaction, 5 mL of deionized water and 1 mL of p-aminobenzenesulfonic acid solution to a 10 ml colorimetric cup in turn, shake well and let stand for 5 minutes to fully react, then add 1 mL of hydrochloric acid and N-(1-naphthyl)-ethylenediamine solution, shake well, dilute to 10 mL with deionized water, read the absorbance value at a wavelength of 540 nm, and do NO in the same way. 2 - The marking line is used to calculate NO 2 – The amount of production is reflected by the nitrite production inhibition rate to reflect the nitrification inhibition effect.

[0033]

[0034] (6) Experimental results are shown in Figure 1 , dihydromicanthone was added at a concentration of 10 μg mL -1 , showing nitrosation inhibition effect, IC 50 (half inhibitory concentration) is 50 μg·mL -1 , E.D. 80 (80% inhibition concentration) is 100 μg·mL -1 At a concentration of 180 μg mL -1 The nitrite inhibition rate reached 100%.

[0035] Example 2 Comparison of the nitrosation ability of dihydromicanthone and DCD nitration inhibitor (dicyandiamide nitration inhibitor).

[0036] (1) Preparation of test solution

[0037] DCD was purchased from Sigma-Aldrich Company, and was dissolved in water to prepare DCD solutions with concentrations of 5 μM, 10 μM, 20 μM, 40 μM, 60 μM, 80 μM, 100 μM, 140 μM, and 180 μM, and stored in a dark place for future use.

[0038] (2) Experimental process

[0039] The method is the same as Example 1, except that the dihydromicanthone solution is replaced by a DCD solution.

[0040] (3) Experimental results are shown in Figure 1

[0041] In the treatments with different addition concentrations, the nitrosation inhibition ability of dihydromicanthone is stronger than that of the most commonly used nitrification inhibitor DCD, and it has considerable nitrosation inhibition ability.

[0042] Example 3 Inhibitory Effects of Dihydromicanthone and DCD on Soil Nitrification Process

[0043] (1) Experimental process

[0044] NH 4 Cl solution was added to fresh soil (brown soil) passed through a 2 mm sieve to provide 400 mgN / kg soil (based on dry soil), then placed in a 100 mL glass culture bottle, treated with deionized water to reach a water holding capacity of 40%, and a DMSO solution of dihydromicanthone and an aqueous solution of DCD were added, each of which was added in an amount of 200 μmol / kg (based on dry soil); placed in a constant temperature incubator at 25°C and cultured at a constant temperature, and soil samples were taken on the 1st, 7th, 14th and 28th days after culture (water was added every day according to the weighing method to keep the total weight of the culture bottle unchanged), and leached with 2 mol potassium chloride solution for 30 minutes of shaking, and the concentrations of ammonium nitrogen and nitrate nitrogen, as well as the inhibition rate of nitrite production were measured, and the steps were the same as in Example 1.

[0045] (2) Experimental results are shown in Figure 2

[0046] Dihydromicanthide can effectively inhibit the nitrosation process in the soil. Within 14 days of soil cultivation, the nitrosation inhibition effect increases with the extension of cultivation time, and the effect is better than that of DCD inhibitor, so it has application prospects.

[0047] Example 4: Inhibitory effects of dihydromicanthone and micanthone on soil nitrification

[0048] (1) Experimental process

[0049] The method is the same as Example 3, except that the DCD solution is replaced by a DMSO solution of micranthol

[0050] (2) Experimental results are shown in Figure 2

[0051] Migania micrantha lactone, which is also a micrantha extract, has a certain ability to inhibit nitrosation in the soil, but its inhibitory ability is not as good as that of dihydromicantha lactone.

[0052] Example 5 Effect of dihydromichanolide on corn yield.

[0053] (1) Experimental process

[0054] Mix 15g of 26-10-12 nitrogen, phosphorus and potassium fertilizers and dihydromicanthone, mix them evenly in 18kg of fresh brown soil as base fertilizer, and do not add dihydromicanthone as CK control. The application rate of dihydromicanthone is 1%, 3% and 5% of the pure nitrogen content, and 5 replicates are set. The nitrogen fertilizer for the test is ammonium sulfate, the phosphorus fertilizer is superphosphate, and the potassium fertilizer is potassium chloride. Then transfer it to a plastic pot (35m in diameter and 35cm in height), and finally adjust the soil moisture content to 60% of the maximum field water holding capacity. Sow 3 corn seeds in each pot, sow 5cm in depth, and thin out the seedlings to one corn plant after emergence (about 15 days). Potted corn is placed in a net room for growth, and artificial watering is carried out every day during the growth period to maintain its soil moisture content. The planting density is about 3,800 plants per mu, and the dry matter weight and yield of corn are measured in autumn.

[0055] (2) Experimental results are shown in Table 1

[0056] Compared with the control treatment with the same nutrients but without dihydromicanthone, the treatment with 0.5% pure nitrogen did not show any increase in dry matter weight and yield. Compared with the control treatment with the same nutrients but without dihydromicanthone, the treatment with 1%, 2% and 3% pure nitrogen significantly increased dry matter weight and yield.

[0057] Table 1 Dry matter mass, yield and yield increase of potted corn under different treatments

[0058]

[0059] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of dihydromicanthide as a nitrification inhibitor.

2. The use according to claim 1, characterized in that: The structural formula of dihydromicanthone is: 。 3. The use according to claim 1, characterized in that: Add 1% to 10% dihydromichanolide by weight of nitrogen to the nitrogen-containing fertilizer.

4. The use according to claim 3, characterized in that: The nitrogen-containing fertilizer is an ammonium nitrogen fertilizer such as ammonium chloride, ammonium sulfate, ammonium bicarbonate, liquid ammonia, monoammonium phosphate or diammonium phosphate.

5. The use according to claim 1, characterized in that: The application method comprises: directly adding dihydromichanolide to nitrogen fertilizer for use.

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