An automated colorimetric method for the determination of urease activity in soil and plant material

Abstract

An automated colorimetric method Cor determining urease activity in soils and plant material is described. The method, using ammonium determination by the nitroprusside‐catalysed indophenol reaction, is Caster and more precise than a colorimetric measurement of urea.     

Short-term assay of soil urease activity using colorimetric determination of ammonium

Summary A rapid assay for soil urease in the absence of bacteriostatic agents has been developed. The method comprises incubation of soil with an aqueous or buffered urea solution, extraction of ammonium with 1 N KCl and 0.01 NHCl and colorimetric NH₄ ⁺ determination by a modified indophenol reaction. The method is characterized by high sensitivity and stability of the coloured complex formed. Measurements obtained by this method showed that no change in urease activity occurred when field-moist samples of soils were stored at –20°C for as long as 5 months. Air-drying of field-moist soil samples may lead to an increase in urease activity.     

Production of urease by microbial activity in soils under aerobic and anaerobic conditions

Summary Several workers have reported that O₂ has little, if any, effect on hydrolysis of urea by soil urease, but others have reported that it has a marked effect, hydrolysis being significantly faster in soils under aerobic conditions than in O₂-depleted soils. In studies to account for these divergent results, we found that whereas plant residues and other readily decomposable organic materials markedly stimulated microbial production of urease in soils under aerobic conditions, they did not greatly stimulate production of urease in soils under anaerobic conditions. We also found that although anaerobic conditions retarded production of urease by soil microorganisms, they did not inhibit hydrolysis of urea by soil urease. These observations suggest that the divergent findings concerning the effect of O₂ on hydrolysis of urea by soil urease may have resulted from differences in the amounts of readily decomposable organic materials in the soils studied.     

Bestimmung der Harnstoffabbaurate in Land- und Unterwasserböden nach verschiedenen Methoden

Determination of urease activity by two different methods in some soils and underwater soils Two methods differing in microbial activity during incubation with urea were used for the determination of urease activity. The results obtained showed a better relationship with natural conditions such as pH, organic substance-, or clay content, if the microbial activity was not inhibited during incubation. This effect was found to be particularly pronounced in underwater soils, where addition of a bacteriostatic substance (toluene) resulted in practically uniform values that reflected neither bacterial nor nutrient status.     

Problems in the use of urea as a nitrogen fertilizer

Abstract. N -( n -butyl) thiophosphoric triamide (NBPT) is the most effective compound currently available for retarding hydrolysis of urea fertilizer in soil and for decreasing ammonia volatilization and nitrite e accumulation in soils treated with urea. It is a poor inhibitor of plant or microbial urease, but decomposes quite rapidly in soil with formation of N -( n -butyl) phosphoric triamide, which is a potent inhibitor of urease activity.
The adverse effects of urea fertilizers on seed germination and seedling growth in soil are due to ammonia produced through hydrolysis of urea by soil urease. They can be eliminated by addition of a urease inhibitor to these fertilizers.
The leaf-burn commonly observed after foliar fertilization of soybeans with urea results from accumulation of toxic amounts of urea in the soybean leaves rather than formation of toxic amounts of ammonia through urea hydrolysis by leaf urease. Leaf-burn is accordingly increased rather than decreased by addition of a urease inhibitor to the urea fertilizer applied.     

Effect of assay conditions and field exposure on urease activity associated with cereal residues

Abstract

Urea fertilisers are often applied to soil with cereal residues on the surface. Although the urease properties of soils have been investigated, the urease activities associated with cereal residues have not been thoroughly studied. Two experiments were performed to develop an acceptable urease assay for cereal residues, and to determine the effect of field exposure and concomitant saprophytic colonization on urease levels.

Two acceptable assays were found: 1) incubation in the presence of THAM‐H₂SO₄ buffer (pH 8), toluene, and urea, and 2) incubation only in the presence of urea. In both assays substrate concentration was 0.05 M, incubation temperature was 37°C, incubation length was 2 hours, and urea hydrolysis was determined by ammonium production. Although both assays were highly correlated (r² = 0.992), the buffer method gave higher values. Subsequent investigations illustrated that urease activity associated with cereal residues is dramatically increased by saprophytic colonization after harvest. Freshly‐harvested winter wheat (Triticum aestivum, L.), winter rye (Secale cereale, L.), and spring wheat residues had urease activities (by the buffer method) of 4.3, 6.9, and 3.1 μmol urea g^‐1 h^‐1, respectively. After field exposure of 67–77 days, the urease activities had increased to 69.1, 101.9, and 74.3, respectively. The latter are far higher than literature values for U.S. mineral soils or the dried green leaves of plants.     

Urea Hydrolysis of Tea Soils as Influenced by Incubation Period,Soil pH,and Nitrification Inhibitor

Abstract

Laboratory incubation studies were conducted with south Indian tea soils to investigate the influence of soil pH, incubation period, and nitrification inhibitor on urea hydrolysis. The soils used in this experiment were sampled from six different regions of south India. The physicochemical properties, urea hydrolysis as influenced by soil pH, incubation period, and nitrification inhibitor were determined. There was a strong positive correlation between urease activity and organic‐matter content of tea soils, whereas physicochemical properties failed to show any relationship with urease activity. The urease activity was highest in Munnar soils. At 25°C, the activity reached maximum within 15 days after fertilizer application, and it was considerably high up to 36 days in the soils of Anamallais, 18 days in Munnar, and 27 days in other zones studied, which revealed the minimum interval between two successive urea fertilizer applications. Soils of different zones showed a different pattern of urease activity when the soil pH was changed artificially between 4 and 5.5. Addition of nitrification inhibitor [dicyandiamide, DCD] to urea prevented nitrate ion formation, resulting in the accumulation of desirable ammonium ions.     

Release and recovery of nitrogen from winter annual cover crops in no‐till corn production

Abstract

Soil bulk density markedly influences hydrolysis of surface‐applied granular urea that is vulnerable to serious ammonia volatilization losses. In order to decrease the ammonia losses by retarding urea hydrolysis, several chemicals have been tested for their soil urease inhibition properties. Phenyl phosphorodiamidate (PPDA) is a potent soil urease inhibitor. Laboratory studies using soil column incubations were conducted to investigate the effect of soil bulk density on inhibition of hydrolysis of surface‐applied urea granules (=20 mg of urea/granule) containing 1% PPDA in unsaturated soils. The increase in soil bulk density (from 0.69 to 1.50 Mg/m³) markedly increased the rate of hydrolysis of surface‐applied urea granules and significantly decreased the apparent urease inhibition by PPDA present in the granules. These results are attributed to the probable spatial separation of urea and PPDA because of the differences in diffusive transports in unsaturated soils caused in part by differences in their solubilities in water.     

Urease activity in two cultivated and non-cultivated arid soils

Summary Since urease activity in soil is believed to be relatively constant, the present study was designed to examine the effects of incubation, soil depth and the effect of cultivation on the persistence of urease activity in arid soils. Two soils were used, a Harkey (coarse, silty, mixed, calcareous, thermic, Typic Torrifluvent) and a Saneli (Clayey over sandy skeletal, montmorillonitic, calcareous, Vertic Torrifluvent), each consisting of a cultivated field and a non-cultivated roadbed site. Urease activity was much lower and more varable in the roadbed soils (40 years without cultivation) than in the cultivated field soils. Pre-incubation for 24 h with urea (with toluene) and without urea (without toluene) greatly reduced the total urease activity in all cases in relation to cell free urease activity (with toluene). Urease activity in the two field soils decreased slightly with profile depth but the decrease was greatest below the plow depth (33 cm). Protease activity or some inactivation processes must have lowered the urease content since there was substantially reduced urease activity after most pre-incubations. The extent of the urease activity decrease was so great that the addition of urea would have been required to increase the production of urease enzyme.     

Urease activity on the phylloplane ‐ measurement and activity levels following urea application

Abstract

Previously employed urea hydrolysis method (4) for the determination urease activity on phylloplanes fails if pre‐applied urea is present as a residue from fertilizer, urine or other sources prior to assay. Under such conditions the total urease activity is underestimated and the extracellular component is overestimated. A modified method is proposed that employs an additional measurement where mercuric chloride is a component of the assay incubating medium, which enables a correction to be made to urease activities estimated under such conditions. However, where no urea is present prior to the assay, previously employed methods are acceptable. Using the proposed modifications, urease activities on the phylloplane which had received urea dressings previously were shown to be depressed over the following 2 or 3 days due to the presence of ammonium ions.     

Effects of substituted p-benzoquinones on urease activity in soils

The effectiveness of substituted p-benzoquinones as inhibitors of urease activity in soils depends largely upon their substituent groups. Methyl, chloro-, bromo- and fluoro-substituted p-benzoquinones have a marked inhibitory effect on soil urease activity, whereas phenyl-, r-butyl-and hydroxy-substituted p-benzoquinones have little, if any, effect. Methyl-substituted p-benzoquinones differ from other substituted p-benzoquinones in that their effects on soil urease activity usually increase markedly with increase in their time of contact with soil.A study of the effects of selected substituted p-benzoquinones on urea hydrolysis and volatilization of urea N as ammonia in a sandy soil treated with urea showed that methyl- and chloro-substituted p-benzoquinones had very marked effects, whereas hydroxy- and phenyl-substituted p-benzoquinones had little or no effect. Gaseous loss of urea N as ammonia when this soil was incubated at 20°C for 14 days was reduced from 62.8 to 0.1 per cent by addition of 2,3-dimethyl-, 2,5-dimethyl-, or 2,6-dimethyl-p-benzoquinone (2.3 parts/100 parts of urea).The work reported indicates that, of the 34 substituted p-benzoquinones studied, 2,3-dimethyl-, 2, 5-dimethyl- and 2,6-dimethyl-p-benzoquinone are likely to prove the most effective for retardation of urea decomposition in soils and reduction of the problems caused by the normally rapid hydrolysis of fertilizer urea by soil urease.     

Persistence of the inhibitory effects of phosphoroamides on urea hydrolysis in soils

Abstract

The persistence of the inhibitory effects of three phosphoroamides [N‐(n‐butyl) thiophosphoric triamide (NBPT), phenylphosphorodiamidate (PPD), and thiophosphoryl triamide (TPT)] on urea hydrolysis in soils was assessed by measuring the ability of four soils to hydrolyze urea after they had been treated with 5 μg phosphoroamide/g soil and incubated at 15°C or 30°C for 0, 3, 7, 14, or 28 days. The soils used differed markedly in pH, texture, and organic‐matter content. The data obtained showed that the persistence of the effects of the phosphoroamides studied decreased with increase in soil temperature from 15°C to 30°C and that whereas the effect of PPD decreased with increase in the time of incubation, the effects of NBPT and TPT sometimes increased before decreasing with increased time of incubation. These observations are in harmony with the recent findings that PPD is a potent inhibitor of urease activity, but decomposes in soils with formation of phenol, which is a relatively weak inhibitor of urease activity, whereas NBPT and TPT do not inhibit urease activity but decompose in soil with formation of their oxon analogs, which are potent inhibitors of urease activity. The inhibitory effect of NBPT on urea hydrolysis was considerably more persistent than that of PPD or TPT and was significant even after incubation of NBPT‐treated soil at 15°C or 30°C for 28 days.     

Production and persistence of urease activity in soils

Addition of urea to Iowa soils did not induce urease activity, but production of urease activity was observed on addition of glucose and other organic materials that promote microbial activity. The persistence of the urease activity produced on addition of these materials varied with the soil, but, with each soil studied, the urease activity after addition of organic materials eventually was identical to that of the unamended soil. No increase or decrease in urease activity was observed when unamended or urea-treated soils were incubated under aerobic conditions for several months. It is concluded that soil constituents protect urease against microbial degradation and other processes leading to inactivation of enzymes and that every soil has a stable level of urease activity determined by the ability of its constituents to provide this protection.     

Soil urease activity and kinetics

The activity and kinetic properties of urease in several Malaysian soils were examined. The values for K_m and V_max of the soils computed according to the Hanes equation were in general agreement with other reports as far as magnitudes were concerned. A significant correlation between K_m and V_max was also obtained. The urease activity of the soils was variable, and it was noted that expression of the activity as the time required to hydrolyze half of the applied urea has limited use in soils of low activity. In all soils studied, inhibition of urease activity was effectively achieved using Ag⁺, while Cu²⁺ was only effective in two soils, and marginally effective in the other two soils. Urease inhibitors have potential applications in reducing volatilization losses of ammonia derived from urea applied to soils.     

Urease activity in some Trinidad soils

The effects of urea concentration, soil moisture content, period of storage of soil samples, temperature and toluene on the urease activity of fifteen Trinidad soils were investigated. From the multiple regression analysis of clay content, organic carbon, CEC and amorphous Fe and Al and the urease activity of the soils, it was found that the urease activity in the presence of toluene was largely associated with the clay-organic matter complex whereas in the absence of toluene it was associated with the organic matter of the soils.     

小叶女贞叶提取物对脲酶的抑制作用

以小叶女贞叶的水提物对脲酶的抑制作用进行研究,目的在于开发一种低廉优质的植物性脲酶抑制剂,同时也是对废弃物的再利用。从小叶女贞的叶片中提取并分离了抑制脲酶的活性部分(LQE),收得率为5.92%。LQE对刀豆脲酶有较强的抑制作用,5 mgLQE与76.3 mg硼砂的抑制效果相当。LQE对脲酶的抑制作用随着温度的升高(4~60℃)而增强。pH5.5~9.0范围内,LQE有明显的抑制脲酶的活性,而且中性和碱性环境中LQE的抑制率高于酸性环境。加入β-巯基乙醇(2-Me)后,脲酶的活性恢复,说明LQE是通过与脲酶的-SH发生作用而导致脲酶的活性降低。LQE可以有效抑制不同土壤中脲酶的活性,延缓尿素的水解,并且其抑制程度随用量的增加而提高。小叶女贞广泛分布于中国,有耐修剪、生长快、低廉、环保等优越性能,因此可以利用其叶片提取物降低土壤脲酶的活性,提高尿素的利用率,减少环境污染。     

Determination of nitrate in soil by second derivative ultraviolet spectrometry

Abstract

A second derivative UV‐spectrometry method is described for the determination of nitrate in soils. Ammonium, nitrite, and urea may be determined in the same extract. The method generally requires no pretreatment of soil extracts, and hence is very rapid. It is largely free from interferences. Recoveries of added nitrate in four soils by the proposed method compared favourably with the phenoldisulphonic acid method.     

尿酶抑制剂氢醌在提高尿素肥效中的作用

用棕壤进行的模拟、盆栽和田间试验的结果表明:1.氢醌对土壤脲酶活性的抑制率随其用量的增多而增大;脲酶活性得以恢复的时间随氢醌用量的减少而缩短。2.适当用量的氢醌能提高春小麦对于尿素氮的利用率和减少尿素氮的气态损失。3.将适当用量的氢醌与尿素于播种前一同施入土中,能显著地提高玉米产量并省去追施氮肥的工序。4.在施有氢醌的处理,不曾发现在土壤、小麦茎秆和籽实中有氢醌的累积。     

关于几种土壤脲酶抑制剂的作用条件

近10年来,几种有效的脲酶抑制剂如N-丁基硫代磷酸三胺N-（n-butyl）thiophosphrictriamide（nBPT）、苯基磷酸二胺phenylphosphorodiamidate（PPD）和氢醌hydroquinone（HQ）受到广泛的关注。脲酶抑制剂能够抑制尿素的水解,因而减少了尿素氮的氨挥发损失。然而,脲酶抑制剂的有效性与环境条件（如土壤pH、通气性以及作物残茬有无）有关。目前的研究表明,脲酶抑制剂用在氨易于挥发的土壤上和脲酶抑制剂的作用环境满足时显示了良好的应用前景。因此应用脲酶抑制剂可能对节省尿素肥料施用量,同时确保提高作物产量、减少环境污染提供一种可持续的农业生产技术。本文论述了几种脲酶抑制剂对尿素氮的转化、作物氮吸收和产量等的影响,使对脲酶抑制剂的潜在农用价值、使用条件及其有效用量有所认识。     

Hydrolysis of urea in two sandy soils under citrus production as influenced by rate and depth of placement

Abstract

The use of urea as a nitrogen (N) source is increasing in citrus production on sandy soils in Florida. Entisol and Spodosol are the two major soil orders used for citrus production. This study was conducted to examine the difference in rate of urea hydrolysis as influenced by depth of placement in a Candler fine sand (Typic Quartzipsamment) and a Wabasso sand (Alfic Haplaquod). These two soils represent the contrasting soils typically found in the citrus growing region of central, southern, and east coast regions of Florida. The rate of urea hydrolysis was faster in a Candler fine sand than that in a Wabasso sand and was greater for the low (0.25 g N/kg) than that for the increased (0.50 or 1.00 g/kg) rates of urea applications in both soils. In a parallel experiment, the rate of urea hydrolysis was examined at various depths (0 to 15‐, 15 to 30‐, 30 to 45‐, and 45 to 60‐cm) in the Candler fine sand using in situ and laboratory incubation studies. The rate of urea hydrolysis decreased with an increase in depth of placement of urea. Increased content of organic matter and higher soil temperature in the surface soil may contribute to increase in urease activity thus resulting in increased rate of urea hydrolysis than that in the lower depth soil.