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Effect of N-(n-butyl) thiophosphori
Effect of N-(n-butyl) thiophosphoric triamide on urea metabolism and the assimilation of ammonium by Triticum aestivum L.
Ekhiñe Artola, Saioa Cruchaga, Idoia Ariz, Jose Fernando Moran, María Garnica, Fabrice Houdusse, José María Garcia Mina, Ignacio Irigoyen, Berta Lasa, Pedro María Aparicio-Tejo
Ekhiñe Artola, Saioa Cruchaga, Idoia Ariz, Jose Fernando Moran, Berta Lasa, Pedro María Aparicio-Tejo
Institute of Agri-Biotechnology, Public University of Navarre- CSIC-Government of Navarre, Campus de Arrosadía s/n, E-31006 Pamplona, Navarra, Spain.
Ignacio Irigoyen
Department of Agrarian Production. Public University of Navarre. Pamplona E-31006, Navarre, Spain.
María Garnica, Fabrice Houdusse, José María Garcia Mina
Departament of R + D, TimacAgro-Grupo Roullier, Polígono Arazuri-Orcoyen, Calle C, Nº32, E-31160, Orcoyen, Navarre, Spain.
Correspondence: Pedro M. Aparicio Tejo
Instituto de Agrobiotecnología. Universidad Pública de Navarra-CSIC-Gobierno de Navarra. Campus de Arrosadía E-31006 Pamplona, Navarra.Spain
Phone: +34 948169122
Fax: +34 948 232191
Email: pmapariciotejo@unavarra.es
http://www.agrobiotecnologia.es/en/grp-fisiologiaMolecular/index.htm
Effect of N-(n-butyl) thiophosphoric triamide on urea metabolism and the assimilation of ammonium by Triticum aestivum L.
Abstract
The use of urea as an N fertilizer has increased to such an extent that it is now the most widely used fertilizer in the world. However, N losses as a result of ammonia volatilization lead to a decrease in its efficiency, therefore different methods have been developed over the years to reduce these losses. One of the most recent involves the use of urea combined with urease inhibitors, such as N-(n-butyl) thiophosphoric triamide (NBPT), in an attempt to delay the hydrolysis of urea in the soil. The aim of this study is to perform an in-depth analysis of the effects that NBPT use has on plant growth and N metabolism.
Wheat plants were cultivated in a greenhouse experiment lasting four weeks and fertilized with urea and NBPT at different concentrations (0, 0.012, 0.062, 0.125%). Each treatment was replicated six times. A non-fertilized control was also cultivated. Several parameters related with N metabolism were analysed at harvest.
NBPT use was found to have visible effects, such as a transitory yellowing of the leaf tips, at the end of the first week of treatment. At a metabolic level, plants treated with the inhibitor were found to have more urea in their tissues and a lower amino acid content, lower glutamine synthetase activity, and lower urease and glutamine synthetase content at the end of the study period, whereas their urease activity seemed to have recovered by this stage.
Keywords: ammonium metabolism; N-(n-butyl) thiophosphoric triamide (NBPT); urea; urease; urease inhibitor; wheat.
Introduction
The widespread contamination arising from agricultural activities is currently one of the most serious environmental problems in many European countries. Indeed, the environmental problems associated with the use of N fertilizers have led to a compromise between achieving increased productivity and a quality harvest, and limiting their environmental impact. “Sustainable” agricultural production requires an in-depth understanding of the processes associated with the transformation of N in the soil and the different ways in which it is absorbed by the plant, and how these factors affect the plant's growth and development. The development of new fertilizers and new methods of application, especially the use of products that can inhibit the activity of those microorganisms associated with transformation of the N present in the soil, is also important.
The use of urea as an N fertilizer has increased to such an extent in the last 25-30 years that nowadays it is the most widely used form of N fertilizer in the world (IFA 2009). The main advantages of this compound are its high N content (46%), its relatively low cost, and its ease of use. However, the efficiency of urea is markedly reduced due to N losses caused by, amongst other factors, ammonia volatilization, which can result in the loss of more than 50% of the N applied (Terman 1979; Bremner 1995). This occurs when urea is hydrolyzed by the enzyme urease released by microorganisms in the soil to form ammonium, which, depending on the pH of the soil, may subsequently be transformed into highly volatile ammonia (Bremner and Krogmeier 1988).
Besides adapting normal agricultural practices for the incorporation of urea into the soil, for example irrigation or by adjusting the dose and application time, several strategies have been developed to reduce the losses resulting from ammonia volatilization. Slow-release fertilizers, where the granules are coated with relatively insoluble substances in order to delay the release of urea, and the application of urease inhibitors, are the most widely used methods in this respect (Trenkel 1997). Urease inhibitors, which have been the subject of numerous studies in the last few years, interact with the active site of the enzyme or a key functional group in the molecule to produce a change in the structure of the active sites, thereby delaying urea hydrolysis and, in turn, promoting the formation of ammonium (Watson 2000). However, a compound has to meet certain requirements, such as lack of toxicity to the plant or the environment, stability, efficiency at low concentrations, compatibility with urea and a price that makes its use economically viable, to be considered a good candidate as an agricultural urease inhibitor. Indeed, although numerous substances have been evaluated, very few have been found to be really effective.
The compounds tested include structural analogs of urea, phosphoramides and thiophosphoramides, and hydroquinones and benzoquinones. N-(n-butyl) thiophosphoric triamide (NBPT) is currently considered to be the most effective means of controlling urea hydrolysis (Bremner and Chai 1986) as its oxidised form, NBPTO, has a higher urease inhibition capacity (Creason et al. 1990). Indeed, the majority of both laboratory and field studies have been carried out with NBPT. This compound has been studied in different species, mainly cereals (Bremner 1995; Malhi et al. 2001; O´Donovan et al. 2008), and has proved to be effective in reducing ammonia volatilization and improving agricultural yields (Bremner and Krogmeier 1988). In fact, it was the first urease inhibitor to be marketed, under the commercial name Agrotain®.
Plants grown with urea and NBPT have been found to have a higher urea content in their tissues (Watson and Miller 1996). This accumulation of urea could be the cause of the phytotoxicity observed in plants grown with urea and urease inhibitor (Krogmeier et al. 1989), and could therefore be a major disadvantage of the use of this product. The majority of this urea is absorbed by the roots, although some of it is endogenous, originating from the different N metabolism processes in the plant. To the best of our knowledge, no studies have yet focussed on the influence of NBPT absorption by plant roots and how this absorption could inhibit plant urease and cause physiological changes in N metabolism. The aim of the present study was therefore to evaluate the effectiveness of NBPT at lower doses than those recommended commercially and to study its effect on the plant's N metabolism.
Materials and methods
Growth conditions
Wheat seeds (Triticum aestivum L.) cv. “Fiel” were sown in 2 L pots containing soil and perlite (1:1 v/v). The soil, with an alkaline pH (8.2), 1.5% organic matter content and sandy loam texture, was obtained from Mendigorría (Navarre, Spain) and was dried in air and sieved beforehand through a 2 mm mesh. A urea solution (180 kg N ha-1) was applied to all pots along with different concentrations of N-(n-butyl) thiophosphoric triamide (0%, 0.012%, 0.062% and 0.125% (w/w)). Additional phosphorous, potassium and magnesium were also applied to the plants at doses of 100, 200 and 50 kg ha-1, respectively. Control pots with no fertilizer applied were also prepared. Each treatment was replicated six times. All 30 pots were placed in a greenhouse, where they remained until the end of the experiment. The plants were irrigated with distilled water up to field capacity during the four weeks that the study lasted whilst ensuring that no drainage occurred.
Plant shoot growth and internal contents
The chlorophyll content (SPAD) and dry weight of plant shoots were measured by placing the leaves in a hot air oven for 48 h at a temperature of 75 ºC. The urea content was determined using a modification of the method described by Witte et al. (2002), and using the colour reagent described by Kyllingsbaek (1975). To avoid possible interference by other molecules in the urea measurement, the samples were previously passed through anion-cation exchange columns (Water Oasis MCX and MAX) to remove any ions that could interfere with the analysis. Extraction of the soluble ions from the tissues was performed in water at 80 ºC. The ammonium concentration was determined using the supernatant obtained after centrifugation, using the phenol-hypochlorite method (Solorzano 1969). Amino acids were extracted into 80% ethanol at 80 ºC. After evaporation of the ethanol, they were re-suspended in water and the amino acid content determined by the ninhydrin reaction using glycine as a standard (Yemm and Cocking 1955). The soluble protein was measured as described by Bradford (1976).
Enzyme activities
Urease activity in the plant tissues was determined by measuring the amount of ammonium formed during the hydrolysis of urea. For the extraction, the sample was ground in liquid N2 and homogenized using 50 mM phosphate buffer at pH 7.5 containing 50 mM NaCl, 1.5% PVPP (w/v), 1 mM EDTA, 2 mM DTT and 4 mM PMSF. After centrifuging the extracts (20 min, 13000g), the supernatants were purified through columns (Sephadex G25-fine). The r
Ekhiñe Artola, Saioa Cruchaga, Idoia Ariz, Jose Fernando Moran, María Garnica, Fabrice Houdusse, José María Garcia Mina, Ignacio Irigoyen, Berta Lasa, Pedro María Aparicio-Tejo
Ekhiñe Artola, Saioa Cruchaga, Idoia Ariz, Jose Fernando Moran, Berta Lasa, Pedro María Aparicio-Tejo
Institute of Agri-Biotechnology, Public University of Navarre- CSIC-Government of Navarre, Campus de Arrosadía s/n, E-31006 Pamplona, Navarra, Spain.
Ignacio Irigoyen
Department of Agrarian Production. Public University of Navarre. Pamplona E-31006, Navarre, Spain.
María Garnica, Fabrice Houdusse, José María Garcia Mina
Departament of R + D, TimacAgro-Grupo Roullier, Polígono Arazuri-Orcoyen, Calle C, Nº32, E-31160, Orcoyen, Navarre, Spain.
Correspondence: Pedro M. Aparicio Tejo
Instituto de Agrobiotecnología. Universidad Pública de Navarra-CSIC-Gobierno de Navarra. Campus de Arrosadía E-31006 Pamplona, Navarra.Spain
Phone: +34 948169122
Fax: +34 948 232191
Email: pmapariciotejo@unavarra.es
http://www.agrobiotecnologia.es/en/grp-fisiologiaMolecular/index.htm
Effect of N-(n-butyl) thiophosphoric triamide on urea metabolism and the assimilation of ammonium by Triticum aestivum L.
Abstract
The use of urea as an N fertilizer has increased to such an extent that it is now the most widely used fertilizer in the world. However, N losses as a result of ammonia volatilization lead to a decrease in its efficiency, therefore different methods have been developed over the years to reduce these losses. One of the most recent involves the use of urea combined with urease inhibitors, such as N-(n-butyl) thiophosphoric triamide (NBPT), in an attempt to delay the hydrolysis of urea in the soil. The aim of this study is to perform an in-depth analysis of the effects that NBPT use has on plant growth and N metabolism.
Wheat plants were cultivated in a greenhouse experiment lasting four weeks and fertilized with urea and NBPT at different concentrations (0, 0.012, 0.062, 0.125%). Each treatment was replicated six times. A non-fertilized control was also cultivated. Several parameters related with N metabolism were analysed at harvest.
NBPT use was found to have visible effects, such as a transitory yellowing of the leaf tips, at the end of the first week of treatment. At a metabolic level, plants treated with the inhibitor were found to have more urea in their tissues and a lower amino acid content, lower glutamine synthetase activity, and lower urease and glutamine synthetase content at the end of the study period, whereas their urease activity seemed to have recovered by this stage.
Keywords: ammonium metabolism; N-(n-butyl) thiophosphoric triamide (NBPT); urea; urease; urease inhibitor; wheat.
Introduction
The widespread contamination arising from agricultural activities is currently one of the most serious environmental problems in many European countries. Indeed, the environmental problems associated with the use of N fertilizers have led to a compromise between achieving increased productivity and a quality harvest, and limiting their environmental impact. “Sustainable” agricultural production requires an in-depth understanding of the processes associated with the transformation of N in the soil and the different ways in which it is absorbed by the plant, and how these factors affect the plant's growth and development. The development of new fertilizers and new methods of application, especially the use of products that can inhibit the activity of those microorganisms associated with transformation of the N present in the soil, is also important.
The use of urea as an N fertilizer has increased to such an extent in the last 25-30 years that nowadays it is the most widely used form of N fertilizer in the world (IFA 2009). The main advantages of this compound are its high N content (46%), its relatively low cost, and its ease of use. However, the efficiency of urea is markedly reduced due to N losses caused by, amongst other factors, ammonia volatilization, which can result in the loss of more than 50% of the N applied (Terman 1979; Bremner 1995). This occurs when urea is hydrolyzed by the enzyme urease released by microorganisms in the soil to form ammonium, which, depending on the pH of the soil, may subsequently be transformed into highly volatile ammonia (Bremner and Krogmeier 1988).
Besides adapting normal agricultural practices for the incorporation of urea into the soil, for example irrigation or by adjusting the dose and application time, several strategies have been developed to reduce the losses resulting from ammonia volatilization. Slow-release fertilizers, where the granules are coated with relatively insoluble substances in order to delay the release of urea, and the application of urease inhibitors, are the most widely used methods in this respect (Trenkel 1997). Urease inhibitors, which have been the subject of numerous studies in the last few years, interact with the active site of the enzyme or a key functional group in the molecule to produce a change in the structure of the active sites, thereby delaying urea hydrolysis and, in turn, promoting the formation of ammonium (Watson 2000). However, a compound has to meet certain requirements, such as lack of toxicity to the plant or the environment, stability, efficiency at low concentrations, compatibility with urea and a price that makes its use economically viable, to be considered a good candidate as an agricultural urease inhibitor. Indeed, although numerous substances have been evaluated, very few have been found to be really effective.
The compounds tested include structural analogs of urea, phosphoramides and thiophosphoramides, and hydroquinones and benzoquinones. N-(n-butyl) thiophosphoric triamide (NBPT) is currently considered to be the most effective means of controlling urea hydrolysis (Bremner and Chai 1986) as its oxidised form, NBPTO, has a higher urease inhibition capacity (Creason et al. 1990). Indeed, the majority of both laboratory and field studies have been carried out with NBPT. This compound has been studied in different species, mainly cereals (Bremner 1995; Malhi et al. 2001; O´Donovan et al. 2008), and has proved to be effective in reducing ammonia volatilization and improving agricultural yields (Bremner and Krogmeier 1988). In fact, it was the first urease inhibitor to be marketed, under the commercial name Agrotain®.
Plants grown with urea and NBPT have been found to have a higher urea content in their tissues (Watson and Miller 1996). This accumulation of urea could be the cause of the phytotoxicity observed in plants grown with urea and urease inhibitor (Krogmeier et al. 1989), and could therefore be a major disadvantage of the use of this product. The majority of this urea is absorbed by the roots, although some of it is endogenous, originating from the different N metabolism processes in the plant. To the best of our knowledge, no studies have yet focussed on the influence of NBPT absorption by plant roots and how this absorption could inhibit plant urease and cause physiological changes in N metabolism. The aim of the present study was therefore to evaluate the effectiveness of NBPT at lower doses than those recommended commercially and to study its effect on the plant's N metabolism.
Materials and methods
Growth conditions
Wheat seeds (Triticum aestivum L.) cv. “Fiel” were sown in 2 L pots containing soil and perlite (1:1 v/v). The soil, with an alkaline pH (8.2), 1.5% organic matter content and sandy loam texture, was obtained from Mendigorría (Navarre, Spain) and was dried in air and sieved beforehand through a 2 mm mesh. A urea solution (180 kg N ha-1) was applied to all pots along with different concentrations of N-(n-butyl) thiophosphoric triamide (0%, 0.012%, 0.062% and 0.125% (w/w)). Additional phosphorous, potassium and magnesium were also applied to the plants at doses of 100, 200 and 50 kg ha-1, respectively. Control pots with no fertilizer applied were also prepared. Each treatment was replicated six times. All 30 pots were placed in a greenhouse, where they remained until the end of the experiment. The plants were irrigated with distilled water up to field capacity during the four weeks that the study lasted whilst ensuring that no drainage occurred.
Plant shoot growth and internal contents
The chlorophyll content (SPAD) and dry weight of plant shoots were measured by placing the leaves in a hot air oven for 48 h at a temperature of 75 ºC. The urea content was determined using a modification of the method described by Witte et al. (2002), and using the colour reagent described by Kyllingsbaek (1975). To avoid possible interference by other molecules in the urea measurement, the samples were previously passed through anion-cation exchange columns (Water Oasis MCX and MAX) to remove any ions that could interfere with the analysis. Extraction of the soluble ions from the tissues was performed in water at 80 ºC. The ammonium concentration was determined using the supernatant obtained after centrifugation, using the phenol-hypochlorite method (Solorzano 1969). Amino acids were extracted into 80% ethanol at 80 ºC. After evaporation of the ethanol, they were re-suspended in water and the amino acid content determined by the ninhydrin reaction using glycine as a standard (Yemm and Cocking 1955). The soluble protein was measured as described by Bradford (1976).
Enzyme activities
Urease activity in the plant tissues was determined by measuring the amount of ammonium formed during the hydrolysis of urea. For the extraction, the sample was ground in liquid N2 and homogenized using 50 mM phosphate buffer at pH 7.5 containing 50 mM NaCl, 1.5% PVPP (w/v), 1 mM EDTA, 2 mM DTT and 4 mM PMSF. After centrifuging the extracts (20 min, 13000g), the supernatants were purified through columns (Sephadex G25-fine). The r
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Tác động của N-(n-butyl) thiophosphoric triamide sự trao đổi chất urê và đồng hóa của amoni bởi Triticum aestivum L.Ekhiñe Artola, Saioa Cruchaga, Idoia Ariz, Jose Fernando Moran, María Garnica, Fabrice Houdusse, José María Garcia Mina, Ignacio Irigoyen, Berta Lasa, Pedro María Aparicio-TejoEkhiñe Artola, Saioa Cruchaga, Idoia Ariz, Jose Fernando Moran, Berta Lasa, Pedro María Aparicio-TejoViện của nông-công nghệ sinh học, khu vực chính phủ đại học Navarre-CSIC của Navarre, Campus de Arrosadía s/n, E-31006 Pamplona, Navarra, Tây Ban Nha.Ignacio IrigoyenVùng nông nghiệp sản xuất. Đại học công cộng của Navarre. Pamplona E-31006, Navarre, Tây Ban Nha.María Garnica, Fabrice Houdusse, José María Garcia MinaDepartament R + D, TimacAgro-Grupo Roullier, Polígono Arazuri-Orcoyen, Calle C, Nº32, E-31160, Orcoyen, Navarre, Tây Ban Nha.Thư từ: Pedro M. Aparicio Tejo Instituto de Agrobiotecnología. Universidad Pública de Navarra CSIC địa phương de Navarra. Khuôn viên trường de Pamplona Arrosadía E-31006, Navarra.SpainĐiện thoại: + 34 948169122Số Fax: + 34 948 232191Thư điện tử: pmapariciotejo@unavarra.eshttp://www.agrobiotecnologia.es/en/GRP-fisiologiaMolecular/index.htm Tác động của N-(n-butyl) thiophosphoric triamide sự trao đổi chất urê và đồng hóa của amoni bởi Triticum aestivum L.Tóm tắtViệc sử dụng urê là một phân bón N đã tăng lên đến mức nó bây giờ là phân bón được sử dụng rộng rãi nhất trên thế giới. Tuy nhiên, N thiệt hại là kết quả của amoniac volatilization dẫn đến sự suy giảm hiệu quả của nó, vì vậy phương pháp khác nhau đã được phát triển trong những năm qua để làm giảm những thiệt hại. Một trong gần đây nhất liên quan đến việc sử dụng của urê kết hợp với chất ức chế urease, chẳng hạn như N-(n-butyl) thiophosphoric triamide (NBPT), trong một nỗ lực để trì hoãn sự thủy phân của urê trong đất. Mục đích của nghiên cứu này là để thực hiện phân tích chuyên sâu của hiệu ứng NBPT sử dụng có trên thực vật tăng trưởng và sự trao đổi chất N. Nhà máy lúa mì được trồng trong một thử nghiệm nhà kính kéo dài 4 tuần và thụ tinh với urê và NBPT ở nồng độ khác nhau (0, 0.012, 0,062, 0,125%). Mỗi lần chữa trị được nhân rộng 6 lần. Một điều khiển không thụ tinh cũng được trồng. Một số thông số liên quan với sự trao đổi chất N đã phân tích lúc thu hoạch.Sử dụng NBPT đã được tìm thấy có tác dụng có thể nhìn thấy, chẳng hạn như một vàng tạm thời của những lời khuyên lá, cuối tuần đầu tiên của điều trị. Ở một mức độ trao đổi chất, nhà máy được điều trị bằng chất ức chế đã được tìm thấy để có thêm urê trong mô của họ và một nội dung axit amin thấp hơn, giảm glutamine synthetase hoạt động, và thấp hơn urease và glutamine synthetase nội dung vào cuối thời gian nghiên cứu, trong khi hoạt động urease của họ dường như đã hồi phục bởi giai đoạn này.Từ khóa: amoni sự trao đổi chất; N-(n-butyl) thiophosphoric triamide (NBPT); urê; urease; chất ức chế urease; lúa mì.Giới thiệuÔ nhiễm phổ biến rộng rãi mà phát sinh từ hoạt động nông nghiệp hiện nay là một trong những vấn đề môi trường nghiêm trọng nhất ở nhiều nước châu Âu. Thật vậy, các vấn đề môi trường kết hợp với việc sử dụng các loại phân bón N đã dẫn tới một sự thỏa hiệp giữa đạt được tăng năng suất và chất lượng thu hoạch một, và hạn chế tác động môi trường của họ. "Bền vững" sản xuất nông nghiệp đòi hỏi sự hiểu biết sâu về các quá trình liên quan đến sự chuyển đổi của N trong đất và những cách khác nhau mà nó được hấp thụ bởi các nhà máy, và làm thế nào những yếu tố ảnh hưởng đến các nhà máy tăng trưởng và phát triển. Sự phát triển mới phân bón và các phương pháp mới của ứng dụng, đặc biệt là việc sử dụng các sản phẩm có thể ức chế hoạt động của các vi sinh vật gắn liền với chuyển đổi của N hiện diện trong đất, cũng là quan trọng. Việc sử dụng urê là một phân bón N đã tăng đến mức 25-30 năm qua mà ngày nay nó là hầu hết các rộng rãi sử dụng các hình thức phân bón N trên thế giới (IFA 2009). Những lợi thế chính của hợp chất này là N nội dung của nó cao có (46%), của nó tương đối rẻ và dễ sử dụng. Tuy nhiên, hiệu quả của urê rõ rệt giảm do N thiệt hại gây ra bởi, trong số các yếu tố khác, volatilization amoniac, mà có thể dẫn đến việc mất hơn 50% N áp dụng (Terman năm 1979; Bremner năm 1995). Điều này xảy ra khi urê thủy phân đạm bởi enzym urease phát hành bởi vi sinh vật trong đất để hình thức amoni, đó, tùy thuộc vào độ pH của đất, có thể sau đó được chuyển đổi thành rất dễ bay hơi amoniac (Bremner và Krogmeier năm 1988). Bên cạnh việc điều chỉnh bình thường thực hành nông nghiệp cho việc kết hợp urê vào đất, ví dụ như thủy lợi hoặc bằng cách điều chỉnh liều và ứng dụng thời gian, một số chiến lược đã được phát triển để giảm các thiệt hại phát sinh từ amoniac volatilization. Phân bón phát hành chậm, nơi các hạt được bọc với các chất không hòa tan tương đối để trì hoãn việc phát hành của urê, và việc áp dụng các chất ức chế urease, là những sử dụng rộng rãi phương pháp trong sự tôn trọng này (Trenkel năm 1997). Ức chế urease, đã là chủ đề của nhiều nghiên cứu trong vài năm qua, tương tác với các trang web hoạt động của enzyme hoặc một nhóm chức quan trọng trong phân tử để sản xuất một sự thay đổi trong cấu trúc của các trang web đang hoạt động, do đó trì hoãn urê thủy phân, và lần lượt, thúc đẩy sự hình thành của amoni (Watson năm 2000). Tuy nhiên, một hợp chất có để đáp ứng các yêu cầu nhất định, chẳng hạn như thiếu của ngộ độc cây hoặc môi trường, sự ổn định, hiệu quả ở nồng độ thấp, khả năng tương thích với urê và một mức giá mà làm cho việc sử dụng nó kinh tế khả thi, để được xem xét một ứng cử viên tốt như là một chất ức chế urease nông nghiệp. Thật vậy, mặc dù nhiều chất đã được đánh giá, rất ít đã được thấy là có hiệu quả thực sự. Các hợp chất thử nghiệm bao gồm cấu trúc chất thay bữa urê, phosphoramides và thiophosphoramides, và hydroquinones và benzoquinones. N-(n-butyl) thiophosphoric triamide (NBPT) hiện nay được coi là hầu hết các phương tiện hiệu quả của việc kiểm soát thủy phân urê (Bremner và Chai 1986) là dạng oxidised, NBPTO, có một khả năng ức chế urease cao (Creason et al. 1990). Thật vậy, phần lớn các lĩnh vực và phòng thí nghiệm nghiên cứu đã được thực hiện với NBPT. Hợp chất này đã được nghiên cứu ở các loài khác nhau, chủ yếu là hạt ngũ cốc (Bremner 1995; Malhi et al. năm 2001; O´Donovan et al 2008), và đã chứng tỏ là có hiệu quả trong việc giảm amoniac volatilization và cải thiện sản lượng nông nghiệp (Bremner và Krogmeier năm 1988). Trong thực tế, nó là chất ức chế urease đầu tiên để được tiếp thị dưới tên thương mại Agrotain ®.Cây trồng với urê và NBPT đã được tìm thấy để có một nội dung urê cao hơn trong các mô của họ (Watson và Miller 1996). Này tích tụ của urê có thể là nguyên nhân gây ra phytotoxicity quan sát thấy ở thực vật phát triển với chất ức chế urê và urease (Krogmeier et al. 1989), và do đó có thể là một bất lợi chính của việc sử dụng của sản phẩm này. Phần lớn các urê này được hấp thụ bởi các rễ, mặc dù một số của nó là nội sinh, có nguồn gốc từ quá trình trao đổi chất khác nhau N trong các nhà máy. Tốt nhất của kiến thức của chúng tôi, không có nghiên cứu đã được tập trung vào sự ảnh hưởng của NBPT hấp thụ bởi rễ cây và làm thế nào hấp thu này có thể ức chế thực vật urease và gây ra thay đổi sinh lý trong sự trao đổi chất N. Mục đích của nghiên cứu hiện nay là do đó để đánh giá hiệu quả của NBPT ở liều thấp hơn so với những người được đề nghị thương mại và để nghiên cứu hiệu quả của nó trên sự trao đổi chất của nhà máy N.Vật liệu và phương phápĐiều kiện tăng trưởngHạt giống lúa mì (Triticum aestivum L.) var. "Fiel" đã được gieo trong 2 L chậu có chứa đất và perlit (1:1 v/v). Đất, với một độ pH kiềm (8.2), 1,5% các kết cấu nội dung và cát loam chất hữu cơ, nhận được từ Mendigorría (Navarre, Tây Ban Nha) và được sấy khô trong không khí và sieved trước thông qua một lưới 2 mm. Một giải pháp urê (180 kg N Hà-1) được áp dụng cho tất cả chậu cùng với các nồng độ khác nhau của N-(n-butyl) thiophosphoric triamide (0%, 0.012%, 0,062% và 0,125% (w/w)). Bổ sung phốt pho, kali và magiê cũng đã được áp dụng cho các nhà máy ở liều 100, 200 và 50 kg Hà-1, tương ứng. Kiểm soát chậu với phân bón không có áp dụng cũng được chuẩn bị. Mỗi lần chữa trị được nhân rộng 6 lần. Tất cả 30 chậu được đặt trong một nhà kính, và họ tiếp tục ở cuối của thử nghiệm. Các nhà máy đã được tưới bằng nước cất đến lĩnh vực khả năng trong thời gian bốn tuần nghiên cứu kéo dài trong khi đảm bảo rằng không có hệ thống thoát nước xảy ra. Máy bắn tăng trưởng và nội bộ nội dungNội dung chất diệp lục (S.VII) và trọng lượng khô của thực vật bắn được đo bằng cách đặt các lá trong một lò khí nóng cho 48 h ở nhiệt độ 75 ºC. Nội dung urê đã là xác định bằng cách sử dụng một biến thể của phương pháp mô tả bởi Witte et al. (2002), và bằng cách sử dụng tinh khiết màu được mô tả bởi Kyllingsbaek (1975). Để tránh có thể can thiệp bởi các phân tử khác trong đo lường urê, các mẫu được trước đó thông qua thông qua anion-cation trao đổi cột (nước ốc đảo MCX và MAX) để loại bỏ bất kỳ các ion có thể ảnh hưởng đến việc phân tích. Khai thác các ion hòa tan từ các mô đã được thực hiện trong nước ở 80 ºC. Nồng độ amoni đã được xác định bằng cách sử dụng supernatant thu được sau khi số, bằng cách sử dụng phương pháp phenol-hypoclorit (Solorzano 1969). Axit amin được chiết xuất thành 80% ethanol lúc 80 ºC. Sau khi sự bay hơi của ethanol, họ đã lại bị đình chỉ trong nước và axit amin nội dung được xác định bằng phản ứng ninhydrin bằng cách sử dụng glycine như một tiêu chuẩn (Yemm và Cocking 1955). Protein hòa tan được đo như được mô tả bởi Bradford (1976).Hoạt động enzymeUrease hoạt động trong các mô thực vật đã được xác định bằng cách đo lượng amoni được hình thành trong thủy phân urê. Cho khai thác, mẫu là mặt đất trong chất lỏng N2 và homogenized sử dụng 50 mM phosphat đệm ở pH 7,5 có 50 mM NaCl, 1,5% PVPP (w/v), 1 mM EDTA, 2 mM DTT và 4 mM PMSF. Sau khi centrifuging các chất chiết xuất (20 phút, 13000g), các supernatants đã được tinh chế thông qua cột (Sephadex G25-Mỹ). R
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Effect of N-(n-butyl) thiophosphoric triamide on urea metabolism and the assimilation of ammonium by Triticum aestivum L.
Ekhiñe Artola, Saioa Cruchaga, Idoia Ariz, Jose Fernando Moran, María Garnica, Fabrice Houdusse, José María Garcia Mina, Ignacio Irigoyen, Berta Lasa, Pedro María Aparicio-Tejo
Ekhiñe Artola, Saioa Cruchaga, Idoia Ariz, Jose Fernando Moran, Berta Lasa, Pedro María Aparicio-Tejo
Institute of Agri-Biotechnology, Public University of Navarre- CSIC-Government of Navarre, Campus de Arrosadía s/n, E-31006 Pamplona, Navarra, Spain.
Ignacio Irigoyen
Department of Agrarian Production. Public University of Navarre. Pamplona E-31006, Navarre, Spain.
María Garnica, Fabrice Houdusse, José María Garcia Mina
Departament of R + D, TimacAgro-Grupo Roullier, Polígono Arazuri-Orcoyen, Calle C, Nº32, E-31160, Orcoyen, Navarre, Spain.
Correspondence: Pedro M. Aparicio Tejo
Instituto de Agrobiotecnología. Universidad Pública de Navarra-CSIC-Gobierno de Navarra. Campus de Arrosadía E-31006 Pamplona, Navarra.Spain
Phone: +34 948169122
Fax: +34 948 232191
Email: pmapariciotejo@unavarra.es
http://www.agrobiotecnologia.es/en/grp-fisiologiaMolecular/index.htm
Effect of N-(n-butyl) thiophosphoric triamide on urea metabolism and the assimilation of ammonium by Triticum aestivum L.
Abstract
The use of urea as an N fertilizer has increased to such an extent that it is now the most widely used fertilizer in the world. However, N losses as a result of ammonia volatilization lead to a decrease in its efficiency, therefore different methods have been developed over the years to reduce these losses. One of the most recent involves the use of urea combined with urease inhibitors, such as N-(n-butyl) thiophosphoric triamide (NBPT), in an attempt to delay the hydrolysis of urea in the soil. The aim of this study is to perform an in-depth analysis of the effects that NBPT use has on plant growth and N metabolism.
Wheat plants were cultivated in a greenhouse experiment lasting four weeks and fertilized with urea and NBPT at different concentrations (0, 0.012, 0.062, 0.125%). Each treatment was replicated six times. A non-fertilized control was also cultivated. Several parameters related with N metabolism were analysed at harvest.
NBPT use was found to have visible effects, such as a transitory yellowing of the leaf tips, at the end of the first week of treatment. At a metabolic level, plants treated with the inhibitor were found to have more urea in their tissues and a lower amino acid content, lower glutamine synthetase activity, and lower urease and glutamine synthetase content at the end of the study period, whereas their urease activity seemed to have recovered by this stage.
Keywords: ammonium metabolism; N-(n-butyl) thiophosphoric triamide (NBPT); urea; urease; urease inhibitor; wheat.
Introduction
The widespread contamination arising from agricultural activities is currently one of the most serious environmental problems in many European countries. Indeed, the environmental problems associated with the use of N fertilizers have led to a compromise between achieving increased productivity and a quality harvest, and limiting their environmental impact. “Sustainable” agricultural production requires an in-depth understanding of the processes associated with the transformation of N in the soil and the different ways in which it is absorbed by the plant, and how these factors affect the plant's growth and development. The development of new fertilizers and new methods of application, especially the use of products that can inhibit the activity of those microorganisms associated with transformation of the N present in the soil, is also important.
The use of urea as an N fertilizer has increased to such an extent in the last 25-30 years that nowadays it is the most widely used form of N fertilizer in the world (IFA 2009). The main advantages of this compound are its high N content (46%), its relatively low cost, and its ease of use. However, the efficiency of urea is markedly reduced due to N losses caused by, amongst other factors, ammonia volatilization, which can result in the loss of more than 50% of the N applied (Terman 1979; Bremner 1995). This occurs when urea is hydrolyzed by the enzyme urease released by microorganisms in the soil to form ammonium, which, depending on the pH of the soil, may subsequently be transformed into highly volatile ammonia (Bremner and Krogmeier 1988).
Besides adapting normal agricultural practices for the incorporation of urea into the soil, for example irrigation or by adjusting the dose and application time, several strategies have been developed to reduce the losses resulting from ammonia volatilization. Slow-release fertilizers, where the granules are coated with relatively insoluble substances in order to delay the release of urea, and the application of urease inhibitors, are the most widely used methods in this respect (Trenkel 1997). Urease inhibitors, which have been the subject of numerous studies in the last few years, interact with the active site of the enzyme or a key functional group in the molecule to produce a change in the structure of the active sites, thereby delaying urea hydrolysis and, in turn, promoting the formation of ammonium (Watson 2000). However, a compound has to meet certain requirements, such as lack of toxicity to the plant or the environment, stability, efficiency at low concentrations, compatibility with urea and a price that makes its use economically viable, to be considered a good candidate as an agricultural urease inhibitor. Indeed, although numerous substances have been evaluated, very few have been found to be really effective.
The compounds tested include structural analogs of urea, phosphoramides and thiophosphoramides, and hydroquinones and benzoquinones. N-(n-butyl) thiophosphoric triamide (NBPT) is currently considered to be the most effective means of controlling urea hydrolysis (Bremner and Chai 1986) as its oxidised form, NBPTO, has a higher urease inhibition capacity (Creason et al. 1990). Indeed, the majority of both laboratory and field studies have been carried out with NBPT. This compound has been studied in different species, mainly cereals (Bremner 1995; Malhi et al. 2001; O´Donovan et al. 2008), and has proved to be effective in reducing ammonia volatilization and improving agricultural yields (Bremner and Krogmeier 1988). In fact, it was the first urease inhibitor to be marketed, under the commercial name Agrotain®.
Plants grown with urea and NBPT have been found to have a higher urea content in their tissues (Watson and Miller 1996). This accumulation of urea could be the cause of the phytotoxicity observed in plants grown with urea and urease inhibitor (Krogmeier et al. 1989), and could therefore be a major disadvantage of the use of this product. The majority of this urea is absorbed by the roots, although some of it is endogenous, originating from the different N metabolism processes in the plant. To the best of our knowledge, no studies have yet focussed on the influence of NBPT absorption by plant roots and how this absorption could inhibit plant urease and cause physiological changes in N metabolism. The aim of the present study was therefore to evaluate the effectiveness of NBPT at lower doses than those recommended commercially and to study its effect on the plant's N metabolism.
Materials and methods
Growth conditions
Wheat seeds (Triticum aestivum L.) cv. “Fiel” were sown in 2 L pots containing soil and perlite (1:1 v/v). The soil, with an alkaline pH (8.2), 1.5% organic matter content and sandy loam texture, was obtained from Mendigorría (Navarre, Spain) and was dried in air and sieved beforehand through a 2 mm mesh. A urea solution (180 kg N ha-1) was applied to all pots along with different concentrations of N-(n-butyl) thiophosphoric triamide (0%, 0.012%, 0.062% and 0.125% (w/w)). Additional phosphorous, potassium and magnesium were also applied to the plants at doses of 100, 200 and 50 kg ha-1, respectively. Control pots with no fertilizer applied were also prepared. Each treatment was replicated six times. All 30 pots were placed in a greenhouse, where they remained until the end of the experiment. The plants were irrigated with distilled water up to field capacity during the four weeks that the study lasted whilst ensuring that no drainage occurred.
Plant shoot growth and internal contents
The chlorophyll content (SPAD) and dry weight of plant shoots were measured by placing the leaves in a hot air oven for 48 h at a temperature of 75 ºC. The urea content was determined using a modification of the method described by Witte et al. (2002), and using the colour reagent described by Kyllingsbaek (1975). To avoid possible interference by other molecules in the urea measurement, the samples were previously passed through anion-cation exchange columns (Water Oasis MCX and MAX) to remove any ions that could interfere with the analysis. Extraction of the soluble ions from the tissues was performed in water at 80 ºC. The ammonium concentration was determined using the supernatant obtained after centrifugation, using the phenol-hypochlorite method (Solorzano 1969). Amino acids were extracted into 80% ethanol at 80 ºC. After evaporation of the ethanol, they were re-suspended in water and the amino acid content determined by the ninhydrin reaction using glycine as a standard (Yemm and Cocking 1955). The soluble protein was measured as described by Bradford (1976).
Enzyme activities
Urease activity in the plant tissues was determined by measuring the amount of ammonium formed during the hydrolysis of urea. For the extraction, the sample was ground in liquid N2 and homogenized using 50 mM phosphate buffer at pH 7.5 containing 50 mM NaCl, 1.5% PVPP (w/v), 1 mM EDTA, 2 mM DTT and 4 mM PMSF. After centrifuging the extracts (20 min, 13000g), the supernatants were purified through columns (Sephadex G25-fine). The r
Ekhiñe Artola, Saioa Cruchaga, Idoia Ariz, Jose Fernando Moran, María Garnica, Fabrice Houdusse, José María Garcia Mina, Ignacio Irigoyen, Berta Lasa, Pedro María Aparicio-Tejo
Ekhiñe Artola, Saioa Cruchaga, Idoia Ariz, Jose Fernando Moran, Berta Lasa, Pedro María Aparicio-Tejo
Institute of Agri-Biotechnology, Public University of Navarre- CSIC-Government of Navarre, Campus de Arrosadía s/n, E-31006 Pamplona, Navarra, Spain.
Ignacio Irigoyen
Department of Agrarian Production. Public University of Navarre. Pamplona E-31006, Navarre, Spain.
María Garnica, Fabrice Houdusse, José María Garcia Mina
Departament of R + D, TimacAgro-Grupo Roullier, Polígono Arazuri-Orcoyen, Calle C, Nº32, E-31160, Orcoyen, Navarre, Spain.
Correspondence: Pedro M. Aparicio Tejo
Instituto de Agrobiotecnología. Universidad Pública de Navarra-CSIC-Gobierno de Navarra. Campus de Arrosadía E-31006 Pamplona, Navarra.Spain
Phone: +34 948169122
Fax: +34 948 232191
Email: pmapariciotejo@unavarra.es
http://www.agrobiotecnologia.es/en/grp-fisiologiaMolecular/index.htm
Effect of N-(n-butyl) thiophosphoric triamide on urea metabolism and the assimilation of ammonium by Triticum aestivum L.
Abstract
The use of urea as an N fertilizer has increased to such an extent that it is now the most widely used fertilizer in the world. However, N losses as a result of ammonia volatilization lead to a decrease in its efficiency, therefore different methods have been developed over the years to reduce these losses. One of the most recent involves the use of urea combined with urease inhibitors, such as N-(n-butyl) thiophosphoric triamide (NBPT), in an attempt to delay the hydrolysis of urea in the soil. The aim of this study is to perform an in-depth analysis of the effects that NBPT use has on plant growth and N metabolism.
Wheat plants were cultivated in a greenhouse experiment lasting four weeks and fertilized with urea and NBPT at different concentrations (0, 0.012, 0.062, 0.125%). Each treatment was replicated six times. A non-fertilized control was also cultivated. Several parameters related with N metabolism were analysed at harvest.
NBPT use was found to have visible effects, such as a transitory yellowing of the leaf tips, at the end of the first week of treatment. At a metabolic level, plants treated with the inhibitor were found to have more urea in their tissues and a lower amino acid content, lower glutamine synthetase activity, and lower urease and glutamine synthetase content at the end of the study period, whereas their urease activity seemed to have recovered by this stage.
Keywords: ammonium metabolism; N-(n-butyl) thiophosphoric triamide (NBPT); urea; urease; urease inhibitor; wheat.
Introduction
The widespread contamination arising from agricultural activities is currently one of the most serious environmental problems in many European countries. Indeed, the environmental problems associated with the use of N fertilizers have led to a compromise between achieving increased productivity and a quality harvest, and limiting their environmental impact. “Sustainable” agricultural production requires an in-depth understanding of the processes associated with the transformation of N in the soil and the different ways in which it is absorbed by the plant, and how these factors affect the plant's growth and development. The development of new fertilizers and new methods of application, especially the use of products that can inhibit the activity of those microorganisms associated with transformation of the N present in the soil, is also important.
The use of urea as an N fertilizer has increased to such an extent in the last 25-30 years that nowadays it is the most widely used form of N fertilizer in the world (IFA 2009). The main advantages of this compound are its high N content (46%), its relatively low cost, and its ease of use. However, the efficiency of urea is markedly reduced due to N losses caused by, amongst other factors, ammonia volatilization, which can result in the loss of more than 50% of the N applied (Terman 1979; Bremner 1995). This occurs when urea is hydrolyzed by the enzyme urease released by microorganisms in the soil to form ammonium, which, depending on the pH of the soil, may subsequently be transformed into highly volatile ammonia (Bremner and Krogmeier 1988).
Besides adapting normal agricultural practices for the incorporation of urea into the soil, for example irrigation or by adjusting the dose and application time, several strategies have been developed to reduce the losses resulting from ammonia volatilization. Slow-release fertilizers, where the granules are coated with relatively insoluble substances in order to delay the release of urea, and the application of urease inhibitors, are the most widely used methods in this respect (Trenkel 1997). Urease inhibitors, which have been the subject of numerous studies in the last few years, interact with the active site of the enzyme or a key functional group in the molecule to produce a change in the structure of the active sites, thereby delaying urea hydrolysis and, in turn, promoting the formation of ammonium (Watson 2000). However, a compound has to meet certain requirements, such as lack of toxicity to the plant or the environment, stability, efficiency at low concentrations, compatibility with urea and a price that makes its use economically viable, to be considered a good candidate as an agricultural urease inhibitor. Indeed, although numerous substances have been evaluated, very few have been found to be really effective.
The compounds tested include structural analogs of urea, phosphoramides and thiophosphoramides, and hydroquinones and benzoquinones. N-(n-butyl) thiophosphoric triamide (NBPT) is currently considered to be the most effective means of controlling urea hydrolysis (Bremner and Chai 1986) as its oxidised form, NBPTO, has a higher urease inhibition capacity (Creason et al. 1990). Indeed, the majority of both laboratory and field studies have been carried out with NBPT. This compound has been studied in different species, mainly cereals (Bremner 1995; Malhi et al. 2001; O´Donovan et al. 2008), and has proved to be effective in reducing ammonia volatilization and improving agricultural yields (Bremner and Krogmeier 1988). In fact, it was the first urease inhibitor to be marketed, under the commercial name Agrotain®.
Plants grown with urea and NBPT have been found to have a higher urea content in their tissues (Watson and Miller 1996). This accumulation of urea could be the cause of the phytotoxicity observed in plants grown with urea and urease inhibitor (Krogmeier et al. 1989), and could therefore be a major disadvantage of the use of this product. The majority of this urea is absorbed by the roots, although some of it is endogenous, originating from the different N metabolism processes in the plant. To the best of our knowledge, no studies have yet focussed on the influence of NBPT absorption by plant roots and how this absorption could inhibit plant urease and cause physiological changes in N metabolism. The aim of the present study was therefore to evaluate the effectiveness of NBPT at lower doses than those recommended commercially and to study its effect on the plant's N metabolism.
Materials and methods
Growth conditions
Wheat seeds (Triticum aestivum L.) cv. “Fiel” were sown in 2 L pots containing soil and perlite (1:1 v/v). The soil, with an alkaline pH (8.2), 1.5% organic matter content and sandy loam texture, was obtained from Mendigorría (Navarre, Spain) and was dried in air and sieved beforehand through a 2 mm mesh. A urea solution (180 kg N ha-1) was applied to all pots along with different concentrations of N-(n-butyl) thiophosphoric triamide (0%, 0.012%, 0.062% and 0.125% (w/w)). Additional phosphorous, potassium and magnesium were also applied to the plants at doses of 100, 200 and 50 kg ha-1, respectively. Control pots with no fertilizer applied were also prepared. Each treatment was replicated six times. All 30 pots were placed in a greenhouse, where they remained until the end of the experiment. The plants were irrigated with distilled water up to field capacity during the four weeks that the study lasted whilst ensuring that no drainage occurred.
Plant shoot growth and internal contents
The chlorophyll content (SPAD) and dry weight of plant shoots were measured by placing the leaves in a hot air oven for 48 h at a temperature of 75 ºC. The urea content was determined using a modification of the method described by Witte et al. (2002), and using the colour reagent described by Kyllingsbaek (1975). To avoid possible interference by other molecules in the urea measurement, the samples were previously passed through anion-cation exchange columns (Water Oasis MCX and MAX) to remove any ions that could interfere with the analysis. Extraction of the soluble ions from the tissues was performed in water at 80 ºC. The ammonium concentration was determined using the supernatant obtained after centrifugation, using the phenol-hypochlorite method (Solorzano 1969). Amino acids were extracted into 80% ethanol at 80 ºC. After evaporation of the ethanol, they were re-suspended in water and the amino acid content determined by the ninhydrin reaction using glycine as a standard (Yemm and Cocking 1955). The soluble protein was measured as described by Bradford (1976).
Enzyme activities
Urease activity in the plant tissues was determined by measuring the amount of ammonium formed during the hydrolysis of urea. For the extraction, the sample was ground in liquid N2 and homogenized using 50 mM phosphate buffer at pH 7.5 containing 50 mM NaCl, 1.5% PVPP (w/v), 1 mM EDTA, 2 mM DTT and 4 mM PMSF. After centrifuging the extracts (20 min, 13000g), the supernatants were purified through columns (Sephadex G25-fine). The r
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- hãy trân trọng những gì mình đang có & đ
- Forerunner of economic globalization
- competent authority
- 216 is the smallest untouchable number w
- The use of urea as an N fertilizer has i
- but ask me to go and pay my late farther
- as soon as
- 216 is the smallest untouchable number w
- Have to of dinner
- English is based on a simple alphabet an
- back in his apartment, he filled his bed
- kích thích ăn vào
- .không có gì thêm để làm nữa
- Bạn có nói được tiếng Việt nam không?
- For
- cho tôi gửi món ăn
- không có gì thêm để làm nữa
- payment
- Have to for dinner
- Trade expansion and openness of trading
- back in his apartment, he filled his bed
- Anh có nói được tiếng Việt nam không?
- GREASE
- finish
