Urease inhibitor and use thereof in crop cultivation

Abstract

A use of trimethylglycine and a salt thereof as a urease inhibitor in enhancing nitrogen fertilizer efficiency. Trimethylglycine and the salt thereof have a highly effective inhibitory effect on urease, and can be used as a nitrogen fertilizer enhancer in crop cultivation.

Description

A urease inhibitor and its application in crop planting TECHNICAL FIELD

[0001] The present application relates to the urease inhibition activity of trimethylglycine and its salts and its application as a 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), resulting in increased fertilizer input costs, reduced output-input ratio, and relatively lower agricultural economic benefits, as well as various ecological environmental problems such as groundwater pollution and increased greenhouse gas emissions.

[0003] Urease in soil is a 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 Sciences, 2018, 51(20): 10) proved that the addition of urease inhibitors can increase the yield of corn, wheat and rice, the three major food crops, 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, 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) have been applied in practice. 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 crystalline prism or leaf, 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 inexpensive.

[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, and can be used as a new type of nitrogen fertilizer synergist in crop planting. SUMMARY

[0007] The present application aims to provide a new high-activity urease inhibitor trimethyl glycine and its salts, which can be used as nitrogen fertilizer synergist in crop planting.

[0008] The urease inhibitor also includes acceptable salts, including hydrochloride, phosphate, citrate, malate, salicylate, fumarate, sulfonate or methanesulfonate.

[0009] A nitrogen fertilizer synergist, which comprises the urease inhibitor or its acceptable salt.

[0010] A nitrogen fertilizer or nitrogen-containing compound fertilizer composition, which refers to a nitrogen-containing fertilizer containing urea or a nitrogen compound fertilizer containing urea, and the composition comprises the urease inhibitor or its acceptable salt, and the proportion of the urease inhibitor in the composition is 0.1wt% to 10wt% of the mass of urea.

[0011] 1. Urease inhibition activity determination

[0012] 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 reacts with urease. The most commonly used test method is the indigo phenol method. First, let the inhibitor react with urease for a period of time, then let it catalyze the decomposition of urea, the generated ammonia gas 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:

[0013] The activity of urease is tested according to the method reported by Weatherburn. The specific steps are as follows: 25μL urease solution (10U / mL) and 25μL test compound (concentration is set according to experiment, prepared with DMSO-phosphate buffer solution) are added to a 96-well plate, incubated at 37℃ for 30min, 50μL urea-containing phosphate buffer solution (25mmol) is added, incubated at 37℃ for 30min, then 50μL A reagent (127mM phenol and 0.168mM sodium nitroprusside) and 50μL B reagent (125mM NaOH and 11.3mM NaOCl) are added, incubated at 37℃ for 30min, and the absorbance value is measured at 620nm. Blank group, normal control group and positive control group (acetylhydroxamic acid) are set, and the inhibition rate and half inhibition concentration IC 50 .

[0014] 2. Field experiment of crops

[0015] Select crop test points, and set up two treatment groups: (1) apply urea; (2) apply urea + urease inhibitor (trimethyl glycine or its salt), the proportion of urease inhibitor added is 0.1% to 10% of the mass of urea. Each treatment is set up with 3 repeats, 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.

[0016] 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 to 5.7 μM, which can be used as a new nitrogen fertilizer synergist in crop planting. DETAILED DESCRIPTION

[0017] Example 1

[0018] In a 96-well plate, add 25 μL of urease (macrobean) solution (10 U / mL), 25 μL of 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 of phosphate buffer solution containing urea 25 mmol, incubate at 37°C for 30 min, then add 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), 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 (acethydroxamic acid), and calculate the half-inhibitory concentration IC 50 of trimethyl glycine on urease, which is 5.4 μM, and the IC 50 of the positive control group acethydroxamic acid is 17.6 μM.

[0019] Example 2

[0020] In a 96-well plate, add 25 μL of urease (macrobean) solution (10 U / mL), 25 μL of trimethyl glycine hydrochloride 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 of phosphate buffer solution containing urea 25 mmol, incubate at 37°C for 30 min, then add 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), 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 (acethydroxamic acid), and calculate the half-inhibitory concentration IC 502.8 μM, positive control group acethydroxamic acid IC 50 17.6 μM.

[0021] Example 3

[0022] In 96-well plates, 25 μL urease (Macrotyloma uniflorum) solution (10 U / mL) and 25 μL trimethylglycine citrate solution (1 μM, 5 μM, 10 μM, 15 μM, 20 μM, 40 μM, prepared with DMSO-phosphate buffer solution) were added, and incubated at 37°C for 30 min. Then, 50 μL phosphate buffer solution containing urea 25 mmol was added, and incubated 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 at 37°C for 30 min. The absorbance value was measured at 620 nm. A blank group, a normal control group, and a positive control group (acethydroxamic acid) were also set up, and the half inhibitory concentration IC50 of trimethylglycine citrate on urease was calculated. 50 2.1 μM, positive control group acethydroxamic acid IC 50 17.6 μM.

[0023] Example 4

[0024] In 96-well plates, 25 μL urease (Macrotyloma uniflorum) solution (10 U / mL) and 25 μL trimethylglycine citrate solution (1 μM, 5 μM, 10 μM, 15 μM, 20 μM, 40 μM, prepared with DMSO-phosphate buffer solution) were added, and incubated at 37°C for 30 min. Then, 50 μL phosphate buffer solution containing urea 25 mmol was added, and incubated 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 at 37°C for 30 min. The absorbance value was measured at 620 nm. A blank group, a normal control group, and a positive control group (acethydroxamic acid) were also set up, and the half inhibitory concentration IC50 of trimethylglycine citrate on urease was calculated. 50 5.7 μM, positive control group acethydroxamic acid IC 50 17.6 μM.

[0025] Example 5

[0026] In 96-well plates, 25 μL urease (Macrotyloma uniflorum) solution (10 U / mL), 25 μL trimethylglycine malate solution (1 μM, 5 μM, 10 μM, 15 μM, 20 μM, 40 μM, prepared with DMSO-phosphate buffer solution), incubated at 37°C for 30 min, 50 μL urea 25 mmol phosphate buffer solution was added, incubated 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, incubated at 37°C for 30 min, and the absorbance value was measured at 620 nm. Blank group, normal control group, positive control group (acetylhydroxamic acid) were set up, and the half inhibitory concentration IC 50 of trimethylglycine malate on urease was calculated as 5.5 μM, and the IC 50 of acetylhydroxamic acid in the positive control group was 17.6 μM.

[0027] Example 6

[0028] A rice 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 + trimethylglycine hydrochloride was applied, and the addition proportion of trimethylglycine hydrochloride was 1% of the mass of urea. Each treatment was set up in triplicate, and the cultivation and fertilization management measures were consistent with the conventional management of local farmers. 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 added trimethylglycine hydrochloride increased by an average of 11.0%.

[0029] Example 7

[0030] 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 + trimethylglycine phosphate was applied, and the addition proportion of trimethylglycine phosphate was 0.8% of the mass of urea. Each treatment was set up in triplicate, and the cultivation and fertilization management measures were consistent with the conventional management of local farmers. 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 added trimethylglycine phosphate increased by an average of 7.6%.

Claims

1. A urease inhibitor, characterized in that, The urease inhibitor is trimethylglycine, and the structural formula of trimethylglycine is:

2. The urease inhibitor of claim 1, characterized in that, The inhibitors include acceptable salts, including hydrochloride, phosphate, citrate, malate, salicylate, fumarate, sulfonate or methanesulfonate.

3. A nitrogen fertilizer synergist characterized in that, The nitrogen fertilizer synergist includes the urease inhibitor of claim 1 or 2.

4. A composition of a nitrogenous fertilizer or a nitrogen-containing compound fertilizer, characterized by, The nitrogen fertilizer or nitrogen compound fertilizer refers to a nitrogen-containing fertilizer containing urea or a nitrogen compound fertilizer containing urea, and the composition contains the urease inhibitor of claim 1 or 2, and the proportion of the urease inhibitor in the composition is 0.1wt% to 10wt% of the mass of urea.

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