A urease inhibitor and its use in the cultivation of crops

Abstract

The present application relates to the application of trimethylglycine and its salts as urease inhibitors in nitrogen fertilizer synergism. Trimethylglycine and its salts have high inhibitory effect on urease, and can be applied as a new type of nitrogen fertilizer synergist in crop planting.

Description

TECHNICAL FIELD

[0001] The present application relates to the urease inhibition activity of trimethylglycine and its salts and the application thereof as nitrogen fertilizer synergist in crop planting. BACKGROUND

[0002] Nitrogen (N) is one of the essential macronutrients for plant growth and development, and plays a key role in crop yield and quality formation. However, the nitrogen fertilizer utilization rate in China is relatively low (Zhou, W. L., Dai, F. Biochemical inhibitor combination on urea nitrogen transformation in yellow clay soil. Journal of Soil and Water Conservation, 2015, 29(5): 95-100), which increases the cost of fertilizer input, reduces the output-input ratio, and relatively reduces the economic benefits of agriculture, while causing various ecological environmental problems such as groundwater pollution and increased greenhouse gas emissions.

[0003] Urease in soil is a kind of hydrolytic enzyme that can catalyze the decomposition of urea into ammonia and carbon dioxide. Inhibition and regulation of urease activity is one of the most effective biochemical methods to improve urea utilization rate. Liu et al. (Liu, Z. H., Wu, X. B., Tan, D. S., et al. Application and environmental effects of one-time fertilization in major food crops in China. Chinese Journal of Agricultural Science, 2018, 51(20): 10) proved that the addition of urease inhibitors can increase the yield of corn, wheat and rice by 5.8% to 22.8%, while significantly reducing ammonia volatilization and nitrous oxide emissions.

[0004] There are more than 100 types of urease inhibitors that have been patented in the world fertilizer market, which are mainly divided into three categories: metal salts, small organic molecules and plant extracts. Currently, only n-butyl thiophosphoric triamide (NBPT), n-propyl thiophosphoryl triamide (NPPT) and hydroquinone (HQ) urease inhibitors have been practically applied. The existing urease inhibitors still have many problems in the use of fertilizers, such as low biological activity, low cost performance, and biological safety.

[0005] Trimethylglycine is a quaternary ammonium alkaloid, and the pure product is a white prismatic or leaf-shaped crystal, which is soluble in water, methanol and ethanol. As a natural plant component, trimethylglycine has the advantages of safety, greenness, non-toxicity and non-pollution, and has been mass-produced and is cheap.

[0006] This study first found that trimethylglycine and its salts have significant urease inhibition activity, and have the advantages of high cost performance and good biological safety, which can be used as a new type of nitrogen fertilizer synergist in crop planting. SUMMARY

[0007] The present application aims to provide a new type of high-activity urease inhibitor, trimethylglycine and its salts, which can be used as a nitrogen fertilizer synergist in crop planting. The structural formula of trimethylglycine is:

[0008] .

[0009] The urease inhibitor also includes acceptable salts, including hydrochloride, phosphate, citrate, malate, salicylate, fumarate, sulfonate or methanesulfonate.

[0010] A nitrogen fertilizer synergist, which comprises the urease inhibitor or the acceptable salt thereof.

[0011] A composition of nitrogen fertilizer or nitrogen-containing compound fertilizer, which refers to nitrogen-containing fertilizer containing urea or nitrogen compound fertilizer containing urea, the composition comprising the urease inhibitor or the acceptable salt thereof, the urease inhibitor accounting for 0.1 wt % to 10 wt % of the mass of urea in the composition.

[0012] 1. Urease inhibition activity assay

[0013] The basic principle of urease inhibitor activity test is to determine the activity of urease inhibitor by detecting the change in the ability of urease to catalyze the decomposition of urea to release ammonia after the urease inhibitor acts on the urease. The most commonly used test method is the indigo phenol method. First, the inhibitor is allowed to react with urease for a period of time, then it is allowed to catalyze the decomposition of urea, the ammonia gas produced is dissolved in buffer, and indigo phenol is used for color development, then the OD value is tested by spectrophotometer, finally the inhibition rate of the inhibitor on urease is calculated, the calculation formula is as follows:

[0014]

[0015] The activity test of urease is carried out according to the method reported by Weatherburn. The specific steps are as follows: 25 μL of urease solution (10 U / mL) and 25 μL of test compound (concentration set according to experiment, prepared with DMSO-phosphate buffer solution) are added to a 96-well plate, incubated at 37°C for 30 min, 50 μL of urea-containing phosphate buffer solution (25 mmol) is added, incubated at 37°C for 30 min, then 50 μL of A reagent (127 mM phenol and 0.168 mM sodium nitroprusside) and 50 μL of B reagent (125 mM NaOH and 11.3 mM NaOCl) are added, incubated at 37°C for 30 min, and the absorbance value is measured at 620 nm. Blank group, normal control group and positive control group (acetylhydroxamic acid) are set, and the inhibition rate and half maximal inhibitory concentration IC 50 .

[0016] 2. Field experiment of crop

[0017] Select crop test points, and set up two treatment groups: (1) apply urea; (2) apply urea + urease inhibitor (trimethyl glycine or its salts), the proportion of urease inhibitor added is 0.1% to 10% of the mass of urea. Each treatment is set up with 3 replicates, and the cultivation and fertilization management measures are consistent with the conventional management of local farmers. The yield is calculated when the crops are harvested.

[0018] The present study found that trimethyl glycine and its salts have significant urease inhibition activity, and the half-inhibitory concentration IC 50 is 4.8 µM-5.7 µM, which can be used as a new nitrogen fertilizer synergist for crop planting. DETAILED DESCRIPTION

[0019] Example 1

[0020] In a 96-well plate, add 25 μL urease (macrohambago) solution (10 U / mL), 25 μL trimethyl glycine solution (1 µM, 5 µM, 10 µM, 15 µM, 20 µM, 40 µM, prepared with DMSO-phosphate buffer solution), incubate at 37°C for 30 min, add 50 μL phosphate buffer solution containing urea 25 mmol, incubate at 37°C for 30 min, then add 50 μL A reagent (127 mM phenol and 0.168 mM sodium nitroprusside) and 50 μL B reagent (125 mM NaOH and 11.3 mM NaOCl), incubate at 37°C for 30 min, and measure the absorbance value at 620 nm. Set up blank group, normal control group, and positive control group (acetylhydroxamic acid), calculate the half-inhibitory concentration IC 50 of trimethyl glycine on urease is 5.4 µM, and the IC 50 of the positive control group acetylhydroxamic acid is 17.6 µM.

[0021] Example 2

[0022] In 96-well plates, 25 μL urease (Macrotyloma uniflorum) solution (10 U / mL) and 25 μL trimethylglycine hydrochloride solution (1 μM, 5 μM, 10 μM, 15 μM, 20 μM, 40 μM, prepared with DMSO-phosphate buffer solution) were incubated together at 37°C for 30 min, 50 μL phosphate buffer solution containing urea 25 mmol was added, and incubated together at 37°C for 30 min, then 50 μL A reagent (127 mM phenol and 0.168 mM sodium nitroprusside) and 50 μL B reagent (125 mM NaOH and 11.3 mM NaOCl) were added, and incubated together at 37°C for 30 min, and the absorbance value was measured at 620 nm. A blank group, a normal control group, and a positive control group (acetyloxime acid) were also set up, and the half-inhibitory concentration IC50of trimethylglycine hydrochloride on urease was calculated 50 was 2.8 μM, and the IC50of the positive control group acetyloxime acid was 17.6 μM. 50

[0023] Example 3

[0024] In 96-well plates, 25 μL urease (Macrotyloma uniflorum) solution (10 U / mL) and 25 μL trimethylglycine phosphate solution (1 μM, 5 μM, 10 μM, 15 μM, 20 μM, 40 μM, prepared with DMSO-phosphate buffer solution) were incubated together at 37°C for 30 min, 50 μL phosphate buffer solution containing urea 25 mmol was added, and incubated together at 37°C for 30 min, then 50 μL A reagent (127 mM phenol and 0.168 mM sodium nitroprusside) and 50 μL B reagent (125 mM NaOH and 11.3 mM NaOCl) were added, and incubated together at 37°C for 30 min, and the absorbance value was measured at 620 nm. A blank group, a normal control group, and a positive control group (acetyloxime acid) were also set up, and the half-inhibitory concentration IC50of trimethylglycine phosphate on urease was calculated 50 was 2.1 μM, and the IC50of the positive control group acetyloxime acid was 17.6 μM. 50

[0025] Example 4

[0026] ​​Add 25 μL of urease (Giant Bean) solution (10 U / mL) and 25 μL of trimethylglycine citrate solution (1 µM, 5 µM, 10 µM, 15 µM, 20 µM, 40 µM, prepared with DMSO-phosphate buffer) to a 96-well plate. Incubate at 37°C for 30 min. Add 50 μL of phosphate buffer containing 25 mmol urea and incubate at 37°C for another 30 min. Then add 50 μL of reagent A (127 mM phenol and 0.168 mM sodium nitroprusside) and 50 μL of reagent B (125 mM NaOH and 11.3 mM NaOCl). Incubate at 37°C for another 30 min and measure the absorbance at 620 nm. Also include a blank control group, a normal control group, and a positive control group (acetyloxyoxime acid). Calculate the half-maximal inhibitory concentration (IC50) of trimethylglycine citrate against urease. 50 The concentration was 5.7 µM, and the IC50 of acetyloxyoxime in the positive control group was 5.7 µM. 50 It is 17.6 µM.

[0027] Example 5

[0028] Add 25 μL of urease (Giant Bean) solution (10 U / mL) and 25 μL of trimethylglycine malate solution (1 µM, 5 µM, 10 µM, 15 µM, 20 µM, 40 µM, prepared with DMSO-phosphate buffer) to a 96-well plate. Incubate at 37°C for 30 min. Add 50 μL of phosphate buffer containing 25 mmol urea and incubate at 37°C for another 30 min. Then add 50 μL of reagent A (127 mM phenol and 0.168 mM sodium nitroprusside) and 50 μL of reagent B (125 mM NaOH and 11.3 mM NaOCl). Incubate at 37°C for another 30 min and measure the absorbance at 620 nm. Also include a blank control group, a normal control group, and a positive control group (acetyloxyoxime acid). Calculate the half-maximal inhibitory concentration (IC50) of trimethylglycine malate against urease. 50 The concentration was 5.5 µM, and the IC50 of acetyloxyoxime in the positive control group was 5.5 µM. 50 It is 17.6 µM.

[0029] Example 6

[0030] A paddy field in Zhijiang, Hubei was selected as the test point, and the rice variety was "Qianliangyou Simiao". Two treatment groups were set up: (1) urea was applied; (2) urea + trimethyl glycine hydrochloride, the addition ratio of trimethyl glycine hydrochloride was 1% of the mass of urea. Each treatment was set up with 3 repeats, and the cultivation and fertilization management measures were consistent with the conventional management of local farmers. The yield was calculated when the rice was harvested: the average yield per mu of group 1 was 1236 Jin, and the average yield per mu of group 2 was 1372 Jin. The yield of rice with the addition of trimethyl glycine hydrochloride increased by 11.0% on average.

[0031] Example 7

[0032] A wheat field in Luohe, Henan was selected as the test point, and the wheat variety was "Zhengmai 1860". Two treatment groups were set up: (1) urea was applied; (2) urea + trimethyl glycine phosphate, the addition ratio of trimethyl glycine phosphate was 0.8% of the mass of urea. Each treatment was set up with 3 repeats, and the cultivation and fertilization management measures were consistent with the conventional management of local farmers. The yield was calculated when the wheat was harvested: the average yield per mu of group 1 was 1566 Jin, and the average yield per mu of group 2 was 1685 Jin. The yield of wheat with the addition of trimethyl glycine phosphate increased by 7.6% on average.

Claims

1. The application of trimethylglycine in urease inhibitors, characterized in that, The structural formula of trimethylglycine is: 。 2. The application according to claim 1, characterized in that, The inhibitors include acceptable salts, including hydrochloride, phosphate, citrate, malate, salicylate, fumarate, sulfonate, or methanesulfonate.

3. The application of trimethylglycine as a urease inhibitor in nitrogen fertilizer or nitrogen-containing compound fertilizer compositions, characterized in that, The nitrogen fertilizer or nitrogen-containing compound fertilizer refers to a nitrogen-containing fertilizer or a nitrogen-containing compound fertilizer containing urea. The composition contains the urease inhibitor as described in claim 1 or 2, and the proportion of the urease inhibitor in the composition is from 0.1 wt% to 10 wt% of the urea mass.

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