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Effects of nitrogen fertilization upon the content of essential amino acids in head chicory (Cichorium intybus L. var. foliosum)
Scientia Horticulturae 92 (2002) 205±215
Effects of nitrogen fertilization upon the content of essential amino acids in head chicory (Cichorium intybus L. var. foliosum) Mirjana CÂusticÂa,*, M. HorvaticÂb, A. Butoracc a
Faculty of Agriculture, Department of Plant Nutrition, University of Zagreb, SvetosÆimunska Cesta 25, HR-10 000 Zagreb, Croatia b Faculty of Pharmacy and Biochemistry, Department of Food Chemistry, University of Zagreb, A. KovacÆicÂa 1, HR-10 000 Zagreb, Croatia c Faculty of Agriculture, Department of General Agronomy, University of Zagreb, SvetosÆimunska Cesta 25, HR-10 000 Zagreb, Croatia Accepted 21 May 2001
Abstract Total nitrogen and essential amino acids in red head chicory were monitored in relation to nitrogen fertilizer rates. Head chicory (Cichorium intybus L. var. foliosum) was grown in a 3-year ®eld trial involving different nitrogen rates (0, 100, 200 kg N ha 1) while potassium and phosphorus fertilization was uniform in all three trial treatments (100 kg K2O ha 1 and 100 kg P2O5 ha 1). Total nitrogen increased under the in¯uence of nitrogen fertilizer and a signi®cant increase P < 0:05 was recorded at the higher fertilizer rate. Plant response to nitrogen fertilizer was manifested differently in the contents of particular essential amino acids both in chicory dry matter and in proteins. A signi®cant decrease P < 0:05 of methionine, valine and lysine contents was determined. The relative decrease, in comparison with the control, was much higher in proteins than in dry matter, especially when the higher nitrogen fertilizer rate was applied. Interaction of nitrogen fertilizer rates and climatic conditions was signi®cant P < 0:05. The adverse effect of nitrogen fertilization was most expressed in methionine content. Regardless of the effect of climatic conditions, the correlation between the relative decrease of this amino acid in proteins and the nitrogen rate applied was highly signi®cant r 0:919; P < 0:01. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Nitrogen fertilization; Nitrogen; Essential amino acids; Head chicory
* Corresponding author. Tel.: 385-41-2393-955; fax: 385-41-2393-605. Â usticÂ). E-mail address: [email protected] (M. C
0304-4238/02/$ ± see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 2 3 8 ( 0 1 ) 0 0 3 0 3 - X
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1. Introduction Adequate nitrogen fertilization of head chicory (Cichorium intybus L. var. foliosum) is important for chicory yield and quality, and also to preserve a clean environment, since excessively high nitrogen fertilizer rates lead to nitrate leaching (Davies and SylvesterBradley, 1995). This is not surprising, since plants generally take up only about 50% of applied nitrogen (Newbould, 1989). Different reports on the effects of nitrogen availability on the biosynthesis of plant amino acids are found in the literature. Most data point to an increase in amino acids, as well as in the biological value of proteins, with nitrogen level in the soil increased up to the optimal value, whereas its further increase leads to an abrupt depression of amino acids (Finck, 1982). It is also known that agroecological conditions as well as plant genetic characteristics have a strong in¯uence upon nitrogen uptake and the chemical composition of plants (Maynard et al., 1976). Most plants are not capable of reducing a high content of soil nitrates and incorporating them into amino compounds, especially if there is not enough light. So surplus nitrate often accumulates within the plants (Elia et al., 1998; Santamaria et al., 1997a; Santamaria and Elia, 1997), becoming a limiting factor for nitrogen metabolism, or it is stored in the form of amides (Mengel and Kirkby, 1987; Marschner, 1995; Whitehead, 1995). Schuphan (1961) recorded a decrease in protein quality with intensi®ed nitrogen application to leafy vegetables, accompanied by a simultaneous rise in nitrates. MuÈller and Hippe (1987) reported that the highest nitrogen rates reduced the content of certain amino acids, at the same time raising the level of nitrates and dry matter in lettuce. Eppendorfer and Bille Sùren (1996) conducted similar research on different vegetable species. Increased nitrogen application leads to a rise in nitrate nitrogen and total nitrogen and a decrease in some amino acids, such as threonine, valine and methionine in spinach and kale. Besides decreasing the quality of proteins, nitrates are also potentially toxic substances for humans (Walker, 1990). Head chicory is one of the less studied species of leafy vegetables, and the objective of this research was to investigate the effect of nitrogen fertilization on the content of essential amino acids in chicory. 2. Materials and methods Research was carried out at the experimental facility of the Faculty of Agriculture in Zagreb, Croatia, in the period from 1991 to 1993. The Maksimir location, where the trial was set-up, has humid climate. The main climate characteristics of the region in which ®eld experiments were carried out, including meteorological conditions in the course of the investigation period, are given in Table 1. It should be pointed out that there was a lack of precipitation at the beginning of the second head chicory vegetation year and a surplus in the third trial year. However, the precipitation recorded during the growing season in these 2 years was higher than the 30-year average. During the research period, August temperatures were higher than the 30-year average in all three research years, while September and October temperatures were more or less within the range common for this region. The head chicory fertilization trial was set-up on hortisol, of pH 6.5 and humus
Year
January
February
Mean air temperature (8C) 1963±1992 0.6 1991 1.8 1992 1.8 1993 1.3 Precipitation (mm) 1963±1992 44 1991 44 1992 10 1993 3 a b
1.8 1.9 4.1 0.6 41 29 40 4
Indicates year average. Indicates total for the year.
March 6.1 8.7 6.6 5.7 57 34 105 22
April 10.5 9.1 11.8 11.9 59 46 28 65
May 15.3 12.4 16.2 18.4 75 100 22 17
June
July
August
18.6 19.1 19.7 19.8
20.4 22.2 21.5 20.9
19.5 20.4 24.4 21.1
98 48 85 86
79 68 59 42
96 61 14 113
September 15.8 17.2 17.4 15.7 78 68 35 150
October 10.4 9.3 10.5 12.0 74 82 199 137
November 5.3 6.1 7.1 2.3 78 140 112 162
December 1.1 1.3 1.4 1.9 57 11 61 104
10.3a 11.9a 10.3a 11.3a 836b 770b 734b 903b
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Table 1 Climatic characteristicsÐmeteorological station, Maksimir, Zagreb
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208
content of ca. 2.1%, in four replications. Three nitrogen rates were applied, a control without nitrogen application, and treatments with 100 and 200 kg N ha 1, while phosphorus and potassium fertilization was uniform in all three trial treatments (100 kg P2O5 ha 1 and 100 kg K2O ha 1). Chicory seed (Cesare, Bejo Zaden) was sown in polystyrene containers and the developed seedlings were planted on the trial area at a 40 30 cm2 spacing at the optimal time for this crop (the ®rst decade of August). The usual standard agricultural engineering practices were applied, i.e. ploughing, preplanting soil preparation by Rototiller, including mineral fertilizer application. From every plot, edible parts of four plants (ca. 2 kg) were taken in a random manner for analyses when the chicory reached maturity (13 and 14 October 1991 and 1993, and 17 November 1992). Total nitrogen (including nitrates) was determined by the modi®ed Kjeldahl method using phenol sulfonic acid (AOAC, 1975), while the xylenol method was applied for nitrates (AOAC, 1975). Amino acids were determined by high performance liquid chromatography (HPLC) after the acid hydrolysis (500 mg of dried homogenized samples of chicory with 6 M HCl, 24 h at 105 8C) (Krishnamurti et al., 1984) using an HPLC apparatus, Milton±Roy type, with ¯uorescence detection. All analyses were made in triplicate, and the results are presented as mean values. The nitrogen to protein conversion factor of 6.25 was used. Statistical data processing was performed with the general linear model (proc. GLM) using the SAS System Software (SAS, 1989). A combined analysis over the years was performed for all traits (measured valuables) (Steel and Torrie, 1980). The linear model for the analysis was Yijk m yi r yj i tk ytik eijk where Yijk is the measured variable, m the general mean, yi the year effect, r(y)j(i) the blocking effect over years, tk the treatment effect, ytik the interaction and eijk the random error. The least significant difference (LSD) test was used for comparison of the means between the fertilization levels (fertilization) as well as for the interaction between the year and fertilization year fertilization means. Correlations between the investigated traits were estimated. 3. Results The content of total nitrogen (NO3-nitrogen) in chicory dry matter ranged from 33.05 to 40.70 g kg 1 in the control, and from 35.95 to 45.95 g kg 1 in the fertilized treatments (Table 2). Statistical processing of experimental data revealed a signi®cant interaction P < 0:05 between the nitrogen rate and climatic characteristics during the trial years with total nitrogen (NO3-nitrogen) in chicory. Total nitrogen (NO3-nitrogen) content was not signi®cantly raised, compared to the control, by the application of 100 kg N ha 1, while application of 200 kg N ha 1 resulted in a signi®cant increase in total nitrogen (NO3-nitrogen) in the 2 trial years (1992 and 1993). To get an insight into the ¯uctuation of total non-nitrate nitrogen in chicory as in¯uenced by the nitrogen fertilizer applied, the nitrate content of chicory was determined.
Year
Fertilization Total nitrogen (NO3-nitrogen) (g kg 1 dry matter) 0 kg N ha
1
100 kg N ha
1
200 kg N ha
NO3-nitrogen (g kg 1
0 kg N ha
1
1
dry matter)
100 kg N ha
1
200 kg N ha
Total nitrogen (g kg 1
0 kg N ha
1
1
dry matter)
100 kg N ha
1
200 kg N ha
1991 1992 1993
33.05 c 33.33 c 40.70 b
36.88 bc 36.75 bc 38.38 bc
35.95 bc 45.33 a 45.95 a
3.51 ab 2.79 c 4.75 a
4.55 ab 3.24 bc 4.27 ab
3.36 ab 4.54 ab 4.52 ab
29.54 c 30.53 bc 35.95 b
32.33 bc 33.51 bc 34.11 bc
32.59 bc 40.78 a 41.43 a
Mean
35.69 b
37.33 b
42.41 a
3.68 a
4.02 a
4.14 a
32.01 b
33.31 b
38.27 a
LSDb (fertilization, P < 0:05) LSDb (year fertilization; P < 0:05) a b
3.38
0.86
3.56
5.86
1.49
6.16
Values of particular components followed by the different letters are significantly different at the 95% confidence level. Least significant difference.
1
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Table 2 Total nitrogen (NO3-nitrogen) and total nitrogen content in chicory according to treatments and yearsa
209
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The correlation between total nitrogen (NO3-nitrogen) and NO3-nitrogen in chicory dry matter was positive and signi®cant (r 0:746, P < 0:05). Correction of total nitrogen (NO3-nitrogen) by the relevant value of NO3-nitrogen (Table 2) gave the value of total non-nitrate nitrogen, in further text total nitrogen (Table 2). Levels of total nitrogen ranged from 29.54 to 35.95 g kg 1 of dry matter in unfertilized controls, and from 32.33 to 41.43 g kg 1 in fertilized treatments, and they also correlated positively and signi®cantly with the content of NO3-nitrogen (r 0:664, P < 0:05). Total nitrogen showed an upward trend under the in¯uence of nitrogen fertilizer, which was not signi®cant at the fertilizer rate of 100 kg N ha 1. Application of 200 kg N ha 1 resulted in a signi®cant increase P < 0:05 of total nitrogen in chicory dry matter in 2 years (1992 and 1993). Data of the amino acid composition analysis indicate that the content of particular essential amino acids, expressed on the dry matter basis, vary under the in¯uence of nitrogen fertilizer application. Statistically signi®cant changes P < 0:05 were determined for methionine, valine and lysine (Tables 3±5). Contents of methionine and valine in chicory dry matter were signi®cantly lower P < 0:05 in both fertilized treatments, 100 and 200 kg N ha 1, than in the control, in all trial years. The correlation between these amino acids and total nitrogen in chicory dry matter was negative and signi®cant (rmethionine 0:669, rvaline 0:681, P < 0:05). A relative decrease of lysine content in chicory dry matter was signi®cant P < 0:05 at the higher nitrogen rate (200 kg N ha 1). The correlation between lysine and total nitrogen was negative, but not signi®cant (r 0:535, P < 0:05). In addition, it should be mentioned that the relative decrease of the methionine content in dry matter correlated signi®cantly with the applied nitrogen fertilizer rate (r 0:976, P < 0:001). Trends of other essential amino acids showed no regularity with regard to nitrogen fertilizer applied in comparison with the control. Table 3 Methionine content in chicory according to treatments and yearsa Year
Fertilization Methionine (g kg 0 kg N ha
1
1
dry matter)
Methionine (g kg
100 kg N ha 1
200 kg N ha 1
0 kg N ha
1
1
crude protein)
100 kg N ha 1
200 kg N ha 1
1991 1992 1993
7.95 a 8.10 a 8.25 a
6.85 b 6.65 b 6.90 b
5.40 c 5.20 c 5.10 c
43.06 a 42.44 a 36.72 b
33.90 c 31.75 d 32.37 d
26.51 e 20.40 f 19.69 f
Mean
8.10 a
6.80 b
5.23 c
40.74 a
32.67 b
22.20 c
LSDb (fertilization, P < 0:05 ) LSDb (year fertilization; P < 0:05)
0.18
0.84
0.31
1.46
a Values of methionine expressed on the same basis (dry matter or crude protein) followed by the different letters are significantly different at the 95% confidence level. b Least significant difference.
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Table 4 Valine content in chicory according to treatments and yearsa Year
Fertilization Valine (g kg
1
0 kg N ha
1
100 kg N ha 1
200 kg N ha 1
0 kg N ha
1991 1992 1993
10.85 a 10.60 a 8.78 c
9.80 b 7.70 f 8.20 de
8.75 c 7.85 ef 8.50 cd
Mean
10.07 a
8.57 b
8.37 b
LSDb (fertilization, P < 0:05 ) LSDb (year fertilization; P < 0:05)
dry matter)
Valine (g kg
1
crude protein) 100 kg N ha 1
200 kg N ha 1
58.76 a 55.55 b 38.94 e
48.50 c 36.76 f 38.47 ef
42.95 d 30.79 h 32.82 g
51.08 a
41.24 b
35.52 c
1
0.24
1.17
0.42
2.03
a Values of valine expressed on the same basis (dry matter or crude protein) followed by the different letters are significantly different at the 95% confidence level. b Least significant difference.
Fluctuation of methionine, valine and lysine content in chicory proteins in relation to nitrogen fertilizer is presented in Tables 3±5. Application of fertilizer at the rate of 100 kg N ha 1 resulted in a signi®cant reduction P < 0:05 of methionine in chicory proteins, in all trial years, and the relative decrease ranged from 13 to 24%. Under the same fertilizing conditions, the contents of valine and lysine were not signi®cantly changed in the year with continuing abundant precipitation (1993). The relative decrease Table 5 Lysine content in chicory according to treatments and yearsa Year
Fertilization Lysine (g kg
1
0 kg N ha
100 kg N ha 1
200 kg N ha 1
0 kg N ha
1
dry matter)
Lysine (g kg 1
1
crude protein)
100 kg N ha 1
200 kg N ha 1
1991 1992 1993
6.50 ab 6.55 a 6.55 a
6.45 abc 6.15 bc 6.30 abc
6.10 c 6.20 abc 6.10 c
35.20 a 34.32 a 29.15 c
31.92 b 29.36 c 29.56 c
29.95 c 24.32 d 23.55 d
Mean
6.53 a
6.30 b
6.13 b
32.89 a
30.28 b
25.94 c
LSDb (fertilization, P < 0:05) LSDb (year fertilization; P < 0:05)
0.21
0.82
0.36
1.42
a Values of lysine expressed on the same basis (dry matter or crude protein) followed by the different letters are significantly different at the 95% confidence level. b Least significant difference.
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ranged from 2 to 33% for valine and 0 to 14% for lysine. The treatment with 200 kg N ha 1 had a statistically signi®cant effect upon the contents of methionine, valine and lysine in proteins. A signi®cant reduction P < 0:05 was determined both relative to the control and to the treatment with 100 kg N ha 1 in all trial years. The relative decrease, compared to the control, ranged from 37 to 52% for methionine, 14 to 45% for valine, and 13 to 27% for lysine. The effect of the interaction between the nitrogen fertilizer rate and climatic conditions in the trial years on the content of these amino acids in chicory proteins was also signi®cant P < 0:05. Regardless of the in¯uence of climatic conditions, there was a positive signi®cant correlation between the relative decrease of the protein methionine content and the applied nitrogen fertilizer rate (r 0:919, P < 0:01). Fluctuation in the content of other essential amino acids, expressed on the crude protein basis, did not show any regularity with respect to nitrogen fertilizer. 4. Discussion The obtained results show that the content of total nitrogen in chicory dry matter is within the limits of that reported in the literature for head lettuce (Lactuca sativa capitata) (Bergmann and Neubert, 1976), lettuce (Lactuca sativa) (Fontes et al., 1997), and head cabbage (Brassica oleracea capitata) (Krug, 1986). Total nitrogen in chicory dry matter was increased signi®cantly by the higher rate of nitrogen fertilizer in the 2 trial years. Those 2 years were characterized by higher precipitation during the growing season, which might have in¯uenced the overall ion mobility and more intensive leaching of NO3-N from the soil, particularly at its higher concentrations (treatment with 200 kg N ha 1), but also lower nitri®cation and thereby a higher uptake of NH4 -N (Haynes, 1986). Since the interaction between NH4 -N and NO3-N in the soil is very complex (Santamaria et al., 1997b) and NH4 -N presence in soil solution may inhibit the uptake of NO3 -N (Haynes and Goh, 1978), the increase of non-nitrate nitrogen in chicory might be a consequence of a rather high uptake of ammonium nitrogen, which was probably accumulated in the amide form. Marschner (1995) maintains that NH4 inhibits the uptake of NO3 if NH4NO3 fertilizers are applied, as it was the case in our research. This might be a consequence of NH4 deprotonation and easier passage through the membrane (Mengel and Kirkby, 1987). The statistically signi®cant increase of total nitrogen in chicory resulting from higher nitrogen rates is in accordance with similar data for endive reported by Santamaria et al. (1997b). As regards the essential amino acid composition, the data reveal that the plant response to the application of nitrogen fertilizer was manifested differently in the contents of particular essential amino acids in chicory dry matter. A signi®cant decrease was determined for methionine as well as for valine and lysine. Changes of the contents of other essential amino acids, with a tendency to decrease, showed no regularity with regard to the nitrogen fertilizer applied. Similar observations were also reported for other leafy vegetables (MuÈller and Hippe, 1987). Further, the interaction of nitrogen fertilizer rate and climatic conditions was signi®cant P < 0:05 relative to the ¯uctuation of essential amino acid levels in chicory
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213
dry matter. This is in agreement with the literature data indicating that the interaction between nitrogen availability and soil water is of utmost importance for protein formation in open cropping (Marschner, 1995). However, regardless of the effect of different climatic conditions in the trial years, methionine content was predominantly affected by the increase of the nitrogen rate; the correlation being highly signi®cant. Comparison between trial years for other essential amino acids shows that the smallest changes in the fertilized treatments were recorded in the year with continuing high precipitation during the growing season. It follows from the data obtained that not essential or non-essential amino acids (CÂusticÂ, 1996), but rather amide nitrogen (Whitehead, 1995) contributed to the increase of total nitrogen in chicory dry matter under the in¯uence of nitrogen fertilizer application. Accumulation of inorganic nitrogen, notably NH3, in leafy vegetables in amide form has been reported by a number of authors (Finck, 1982; Yagodin, 1984; Mengel and Kirkby, 1987). The relation between the contents of particular essential amino acids in proteins was similar to their interrelationship in dry matter in the fertilized treatments. However, the relative decrease of certain amino acids in proteins of fertilized chicory, as compared to the control, was substantially higher than the relative decrease of these amino acids expressed on the dry matter basis. This held particularly for the application of the higher nitrogen rate, and was associated with the increase of total nitrogen, namely crude proteins in fertilized treatments. The correlation between the content of all essential amino acids, expressed on the protein basis, and the content of total nitrogen in chicory dry matter was negative and highly signi®cant (Table 6). This observation is consistent with literature data for some other leafy vegetables (Eppendorfer and Bille Sùren, 1996) and indicates depression of the nutritive quality of chicory proteins in fertilized treatments. The effect of nitrogen fertilizer was most pronounced in the relative decrease of methionine in chicory proteins. This may be due to the depression of methionine biosynthesis or to its increased involvement, as the methyl-group donor, in the methylation processes over the course of plant development in the conditions of the higher nitrogen rate application. The results are in agreement with other investigations that also proved high susceptibility of methionine in leafy vegetables connected with the increased Table 6 Correlation coefficients between essential amino acids in proteins and total nitrogen in chicory dry matter r Methionine Valine Lysine Leucine Isoleucine Phenylalanine Threonine **
r 0:01 n 2 0:750. r 0:001 n 2 0:875.
***
0.853** 0.864** 0.974*** 0.831** 0.882*** 0.783** 0.813**
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nitrogen application. Schuphan (1961) reported a signi®cant decrease of methionine content in spinach proteins and so did Eppendorfer and Bille Sùren (1996) for kale proteins and cauli¯ower. 5. Conclusion The obtained results allow the conclusion that the application of nitrogen fertilizer in the production of red head chicory leads to depression of certain essential amino acids, both in dry matter and proteins, at the same time increasing the total nitrogen content. Methionine, valine and lysine levels decrease signi®cantly, particularly with the application of 200 kg N ha 1, where the interaction of the nitrogen rate and weather conditions is signi®cant. It follows from the foregoing that, in the production of red head chicory on hortisol, the nitrogen fertilizer rate supposed to ensure a relatively good nutritive quality of the proteins should not exceed 100 kg N ha 1 in the conditions of optimal humidity. References AOAC, 1975. Methods of Analysis of the Association of Official Analytical Chemists, Washington. Bergmann, W., Neubert, P., 1976. Pflanzendiagnose und Pflanzenanalyse. VEB Gustav Fischer Verlag Jena. CÂusticÂ, M., 1996. Influence of nitrogen fertilization upon the amino acids composition in head chicory. Dissertation, Zagreb. Davies, D.B., Sylvester-Bradley, R., 1995. The contribution of fertiliser nitrogen to leachable nitrogen in the UK: a review. ADAS Soil and Water Research Centre, Cambridge, UK. Elia, A., Santamaria, P., Serio, F., 1998. Nitrogen nutrition, yield and quality of spinach. J. Sci. Food Agric. 76, 341±346. Eppendorfer, W., Bille Sùren, W., 1996. Free and total amino acid composition of edible parts of beans, kale, spinach, cauliflower and potatoes as influenced by nitrogen fertilisation and phosphorus and potassium deficiency. J. Sci. Food Agric. 68, 449±458. Finck, A., 1982. Fertilizers and Fertilization. Introduction and Practical Guide to Crop Fertilization. Weinhei/ Deerfield Beach, Florida/Basel. Fontes, P.C.R., Pereira, P.R.G., Conde, R.M., 1997. Critical chlorophyll, total nitrogen, and nitrate-nitrogen in leaves associated to maximum lettuce yield. J. Plant Nutr. 20, 1061±1068. Haynes, R.J., 1986. Mineral Nitrogen in the Plant±Soil System. Academic Press, Orlando, FL. Haynes, R.J., Goh, K.M., 1978. Ammonium and nitrate nutrition of plants. Biol. Rev. 53, 465±510. Krishnamurti, C.R., Heindze, A.M., Galzy, G., 1984. Application of reversed-phase high-performance liquid chromatography using pre-column derivatisation with o-phthaldialdehyde for the quantitative analysis of amino acids in adult and fetal sheep plasma, animal feeds and tissues. J. Chrom. 315, 321±331. Krug, H., 1986. GemuÈseproduktion, Verlag Paul Parley, Berlin und Hamburg. Marschner, H., 1995. Mineral Nutrition of Higher Plants, 2nd Edition. Academic Press, London. Maynard, D.N., Barker, A.V., Minotti, P.L., Peck, N.H., 1976. Nitrate accumulation in vegetables. Adv. Agron. 28, 71±118. Mengel, K., Kirkby, E.A., 1987. Principles of Plant Nutrition. International Potash Institute, Bern. MuÈller, K., Hippe, J., 1987. Influence of differences in nutrition on important quality characteristics of some agricultural crops. Plant Soil 100, 35±45. Newbould, P., 1989. The use of nitrogen fertiliser in agriculture. Where do we go practically and ecologically. Plant Soil 115, 297±311. Santamaria, P., Elia, A., 1997. Producing nitrate-free endive heads: effect of nitrogen form on growth, yield and ion composition of endive. J. Am. Soc. Hort. Sci. 122, 140±145.
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215
Santamaria, P., Elia, A., Gonnella, M., Serio, F., 1997a. Ways of reducing nitrate content in hydroponically grown leafy vegetables. In: Proceedings of the Ninth International Congress on Soilless Culture, pp. 437±451. Santamaria, P., Elia, A., Gonella, M., 1997b. Changes in nitrate accumulation and growth of endive plants during light period as affected by nitrogen level and form. J. Plant Nutr. 20, 1255±1266. SAS, 1989. SAS/STAT User's Guide, Ver. 6, Vols. 1 and 2, 4th Edition. SAS Institute, Inc., Cary, NC. Schuphan, W., 1961. Methioningehalt und EiweisqualitaÈt von Blattpflanzen in AbhaÈngigkeit von der StickstoffduÈngung. Qual. Plant. Mat. Veg. 8, 261±283. Steel, R.G.D., Torrie, J.H., 1980. Principles and Procedures of Statistics. McGraw-Hill, New York. Walker, R., 1990. Nitrates, nitrites and N-nitroso compounds: a review of the occurrence in food and diet and the toxicological implications. Food Addit. Contam. 6, 717±768. Whitehead, D.C., 1995. Grassland Nitrogen. CAB International, Oxon, UK. Yagodin, B.A., 1984. Agricultural Chemistry. Mir, Moscow.
Effects of nitrogen fertilization upon the content of essential amino acids in head chicory (Cichorium intybus L. var. foliosum) Mirjana CÂusticÂa,*, M. HorvaticÂb, A. Butoracc a
Faculty of Agriculture, Department of Plant Nutrition, University of Zagreb, SvetosÆimunska Cesta 25, HR-10 000 Zagreb, Croatia b Faculty of Pharmacy and Biochemistry, Department of Food Chemistry, University of Zagreb, A. KovacÆicÂa 1, HR-10 000 Zagreb, Croatia c Faculty of Agriculture, Department of General Agronomy, University of Zagreb, SvetosÆimunska Cesta 25, HR-10 000 Zagreb, Croatia Accepted 21 May 2001
Abstract Total nitrogen and essential amino acids in red head chicory were monitored in relation to nitrogen fertilizer rates. Head chicory (Cichorium intybus L. var. foliosum) was grown in a 3-year ®eld trial involving different nitrogen rates (0, 100, 200 kg N ha 1) while potassium and phosphorus fertilization was uniform in all three trial treatments (100 kg K2O ha 1 and 100 kg P2O5 ha 1). Total nitrogen increased under the in¯uence of nitrogen fertilizer and a signi®cant increase P < 0:05 was recorded at the higher fertilizer rate. Plant response to nitrogen fertilizer was manifested differently in the contents of particular essential amino acids both in chicory dry matter and in proteins. A signi®cant decrease P < 0:05 of methionine, valine and lysine contents was determined. The relative decrease, in comparison with the control, was much higher in proteins than in dry matter, especially when the higher nitrogen fertilizer rate was applied. Interaction of nitrogen fertilizer rates and climatic conditions was signi®cant P < 0:05. The adverse effect of nitrogen fertilization was most expressed in methionine content. Regardless of the effect of climatic conditions, the correlation between the relative decrease of this amino acid in proteins and the nitrogen rate applied was highly signi®cant r 0:919; P < 0:01. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Nitrogen fertilization; Nitrogen; Essential amino acids; Head chicory
* Corresponding author. Tel.: 385-41-2393-955; fax: 385-41-2393-605. Â usticÂ). E-mail address: [email protected] (M. C
0304-4238/02/$ ± see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 2 3 8 ( 0 1 ) 0 0 3 0 3 - X
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1. Introduction Adequate nitrogen fertilization of head chicory (Cichorium intybus L. var. foliosum) is important for chicory yield and quality, and also to preserve a clean environment, since excessively high nitrogen fertilizer rates lead to nitrate leaching (Davies and SylvesterBradley, 1995). This is not surprising, since plants generally take up only about 50% of applied nitrogen (Newbould, 1989). Different reports on the effects of nitrogen availability on the biosynthesis of plant amino acids are found in the literature. Most data point to an increase in amino acids, as well as in the biological value of proteins, with nitrogen level in the soil increased up to the optimal value, whereas its further increase leads to an abrupt depression of amino acids (Finck, 1982). It is also known that agroecological conditions as well as plant genetic characteristics have a strong in¯uence upon nitrogen uptake and the chemical composition of plants (Maynard et al., 1976). Most plants are not capable of reducing a high content of soil nitrates and incorporating them into amino compounds, especially if there is not enough light. So surplus nitrate often accumulates within the plants (Elia et al., 1998; Santamaria et al., 1997a; Santamaria and Elia, 1997), becoming a limiting factor for nitrogen metabolism, or it is stored in the form of amides (Mengel and Kirkby, 1987; Marschner, 1995; Whitehead, 1995). Schuphan (1961) recorded a decrease in protein quality with intensi®ed nitrogen application to leafy vegetables, accompanied by a simultaneous rise in nitrates. MuÈller and Hippe (1987) reported that the highest nitrogen rates reduced the content of certain amino acids, at the same time raising the level of nitrates and dry matter in lettuce. Eppendorfer and Bille Sùren (1996) conducted similar research on different vegetable species. Increased nitrogen application leads to a rise in nitrate nitrogen and total nitrogen and a decrease in some amino acids, such as threonine, valine and methionine in spinach and kale. Besides decreasing the quality of proteins, nitrates are also potentially toxic substances for humans (Walker, 1990). Head chicory is one of the less studied species of leafy vegetables, and the objective of this research was to investigate the effect of nitrogen fertilization on the content of essential amino acids in chicory. 2. Materials and methods Research was carried out at the experimental facility of the Faculty of Agriculture in Zagreb, Croatia, in the period from 1991 to 1993. The Maksimir location, where the trial was set-up, has humid climate. The main climate characteristics of the region in which ®eld experiments were carried out, including meteorological conditions in the course of the investigation period, are given in Table 1. It should be pointed out that there was a lack of precipitation at the beginning of the second head chicory vegetation year and a surplus in the third trial year. However, the precipitation recorded during the growing season in these 2 years was higher than the 30-year average. During the research period, August temperatures were higher than the 30-year average in all three research years, while September and October temperatures were more or less within the range common for this region. The head chicory fertilization trial was set-up on hortisol, of pH 6.5 and humus
Year
January
February
Mean air temperature (8C) 1963±1992 0.6 1991 1.8 1992 1.8 1993 1.3 Precipitation (mm) 1963±1992 44 1991 44 1992 10 1993 3 a b
1.8 1.9 4.1 0.6 41 29 40 4
Indicates year average. Indicates total for the year.
March 6.1 8.7 6.6 5.7 57 34 105 22
April 10.5 9.1 11.8 11.9 59 46 28 65
May 15.3 12.4 16.2 18.4 75 100 22 17
June
July
August
18.6 19.1 19.7 19.8
20.4 22.2 21.5 20.9
19.5 20.4 24.4 21.1
98 48 85 86
79 68 59 42
96 61 14 113
September 15.8 17.2 17.4 15.7 78 68 35 150
October 10.4 9.3 10.5 12.0 74 82 199 137
November 5.3 6.1 7.1 2.3 78 140 112 162
December 1.1 1.3 1.4 1.9 57 11 61 104
10.3a 11.9a 10.3a 11.3a 836b 770b 734b 903b
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Table 1 Climatic characteristicsÐmeteorological station, Maksimir, Zagreb
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208
content of ca. 2.1%, in four replications. Three nitrogen rates were applied, a control without nitrogen application, and treatments with 100 and 200 kg N ha 1, while phosphorus and potassium fertilization was uniform in all three trial treatments (100 kg P2O5 ha 1 and 100 kg K2O ha 1). Chicory seed (Cesare, Bejo Zaden) was sown in polystyrene containers and the developed seedlings were planted on the trial area at a 40 30 cm2 spacing at the optimal time for this crop (the ®rst decade of August). The usual standard agricultural engineering practices were applied, i.e. ploughing, preplanting soil preparation by Rototiller, including mineral fertilizer application. From every plot, edible parts of four plants (ca. 2 kg) were taken in a random manner for analyses when the chicory reached maturity (13 and 14 October 1991 and 1993, and 17 November 1992). Total nitrogen (including nitrates) was determined by the modi®ed Kjeldahl method using phenol sulfonic acid (AOAC, 1975), while the xylenol method was applied for nitrates (AOAC, 1975). Amino acids were determined by high performance liquid chromatography (HPLC) after the acid hydrolysis (500 mg of dried homogenized samples of chicory with 6 M HCl, 24 h at 105 8C) (Krishnamurti et al., 1984) using an HPLC apparatus, Milton±Roy type, with ¯uorescence detection. All analyses were made in triplicate, and the results are presented as mean values. The nitrogen to protein conversion factor of 6.25 was used. Statistical data processing was performed with the general linear model (proc. GLM) using the SAS System Software (SAS, 1989). A combined analysis over the years was performed for all traits (measured valuables) (Steel and Torrie, 1980). The linear model for the analysis was Yijk m yi r yj i tk ytik eijk where Yijk is the measured variable, m the general mean, yi the year effect, r(y)j(i) the blocking effect over years, tk the treatment effect, ytik the interaction and eijk the random error. The least significant difference (LSD) test was used for comparison of the means between the fertilization levels (fertilization) as well as for the interaction between the year and fertilization year fertilization means. Correlations between the investigated traits were estimated. 3. Results The content of total nitrogen (NO3-nitrogen) in chicory dry matter ranged from 33.05 to 40.70 g kg 1 in the control, and from 35.95 to 45.95 g kg 1 in the fertilized treatments (Table 2). Statistical processing of experimental data revealed a signi®cant interaction P < 0:05 between the nitrogen rate and climatic characteristics during the trial years with total nitrogen (NO3-nitrogen) in chicory. Total nitrogen (NO3-nitrogen) content was not signi®cantly raised, compared to the control, by the application of 100 kg N ha 1, while application of 200 kg N ha 1 resulted in a signi®cant increase in total nitrogen (NO3-nitrogen) in the 2 trial years (1992 and 1993). To get an insight into the ¯uctuation of total non-nitrate nitrogen in chicory as in¯uenced by the nitrogen fertilizer applied, the nitrate content of chicory was determined.
Year
Fertilization Total nitrogen (NO3-nitrogen) (g kg 1 dry matter) 0 kg N ha
1
100 kg N ha
1
200 kg N ha
NO3-nitrogen (g kg 1
0 kg N ha
1
1
dry matter)
100 kg N ha
1
200 kg N ha
Total nitrogen (g kg 1
0 kg N ha
1
1
dry matter)
100 kg N ha
1
200 kg N ha
1991 1992 1993
33.05 c 33.33 c 40.70 b
36.88 bc 36.75 bc 38.38 bc
35.95 bc 45.33 a 45.95 a
3.51 ab 2.79 c 4.75 a
4.55 ab 3.24 bc 4.27 ab
3.36 ab 4.54 ab 4.52 ab
29.54 c 30.53 bc 35.95 b
32.33 bc 33.51 bc 34.11 bc
32.59 bc 40.78 a 41.43 a
Mean
35.69 b
37.33 b
42.41 a
3.68 a
4.02 a
4.14 a
32.01 b
33.31 b
38.27 a
LSDb (fertilization, P < 0:05) LSDb (year fertilization; P < 0:05) a b
3.38
0.86
3.56
5.86
1.49
6.16
Values of particular components followed by the different letters are significantly different at the 95% confidence level. Least significant difference.
1
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Table 2 Total nitrogen (NO3-nitrogen) and total nitrogen content in chicory according to treatments and yearsa
209
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The correlation between total nitrogen (NO3-nitrogen) and NO3-nitrogen in chicory dry matter was positive and signi®cant (r 0:746, P < 0:05). Correction of total nitrogen (NO3-nitrogen) by the relevant value of NO3-nitrogen (Table 2) gave the value of total non-nitrate nitrogen, in further text total nitrogen (Table 2). Levels of total nitrogen ranged from 29.54 to 35.95 g kg 1 of dry matter in unfertilized controls, and from 32.33 to 41.43 g kg 1 in fertilized treatments, and they also correlated positively and signi®cantly with the content of NO3-nitrogen (r 0:664, P < 0:05). Total nitrogen showed an upward trend under the in¯uence of nitrogen fertilizer, which was not signi®cant at the fertilizer rate of 100 kg N ha 1. Application of 200 kg N ha 1 resulted in a signi®cant increase P < 0:05 of total nitrogen in chicory dry matter in 2 years (1992 and 1993). Data of the amino acid composition analysis indicate that the content of particular essential amino acids, expressed on the dry matter basis, vary under the in¯uence of nitrogen fertilizer application. Statistically signi®cant changes P < 0:05 were determined for methionine, valine and lysine (Tables 3±5). Contents of methionine and valine in chicory dry matter were signi®cantly lower P < 0:05 in both fertilized treatments, 100 and 200 kg N ha 1, than in the control, in all trial years. The correlation between these amino acids and total nitrogen in chicory dry matter was negative and signi®cant (rmethionine 0:669, rvaline 0:681, P < 0:05). A relative decrease of lysine content in chicory dry matter was signi®cant P < 0:05 at the higher nitrogen rate (200 kg N ha 1). The correlation between lysine and total nitrogen was negative, but not signi®cant (r 0:535, P < 0:05). In addition, it should be mentioned that the relative decrease of the methionine content in dry matter correlated signi®cantly with the applied nitrogen fertilizer rate (r 0:976, P < 0:001). Trends of other essential amino acids showed no regularity with regard to nitrogen fertilizer applied in comparison with the control. Table 3 Methionine content in chicory according to treatments and yearsa Year
Fertilization Methionine (g kg 0 kg N ha
1
1
dry matter)
Methionine (g kg
100 kg N ha 1
200 kg N ha 1
0 kg N ha
1
1
crude protein)
100 kg N ha 1
200 kg N ha 1
1991 1992 1993
7.95 a 8.10 a 8.25 a
6.85 b 6.65 b 6.90 b
5.40 c 5.20 c 5.10 c
43.06 a 42.44 a 36.72 b
33.90 c 31.75 d 32.37 d
26.51 e 20.40 f 19.69 f
Mean
8.10 a
6.80 b
5.23 c
40.74 a
32.67 b
22.20 c
LSDb (fertilization, P < 0:05 ) LSDb (year fertilization; P < 0:05)
0.18
0.84
0.31
1.46
a Values of methionine expressed on the same basis (dry matter or crude protein) followed by the different letters are significantly different at the 95% confidence level. b Least significant difference.
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Table 4 Valine content in chicory according to treatments and yearsa Year
Fertilization Valine (g kg
1
0 kg N ha
1
100 kg N ha 1
200 kg N ha 1
0 kg N ha
1991 1992 1993
10.85 a 10.60 a 8.78 c
9.80 b 7.70 f 8.20 de
8.75 c 7.85 ef 8.50 cd
Mean
10.07 a
8.57 b
8.37 b
LSDb (fertilization, P < 0:05 ) LSDb (year fertilization; P < 0:05)
dry matter)
Valine (g kg
1
crude protein) 100 kg N ha 1
200 kg N ha 1
58.76 a 55.55 b 38.94 e
48.50 c 36.76 f 38.47 ef
42.95 d 30.79 h 32.82 g
51.08 a
41.24 b
35.52 c
1
0.24
1.17
0.42
2.03
a Values of valine expressed on the same basis (dry matter or crude protein) followed by the different letters are significantly different at the 95% confidence level. b Least significant difference.
Fluctuation of methionine, valine and lysine content in chicory proteins in relation to nitrogen fertilizer is presented in Tables 3±5. Application of fertilizer at the rate of 100 kg N ha 1 resulted in a signi®cant reduction P < 0:05 of methionine in chicory proteins, in all trial years, and the relative decrease ranged from 13 to 24%. Under the same fertilizing conditions, the contents of valine and lysine were not signi®cantly changed in the year with continuing abundant precipitation (1993). The relative decrease Table 5 Lysine content in chicory according to treatments and yearsa Year
Fertilization Lysine (g kg
1
0 kg N ha
100 kg N ha 1
200 kg N ha 1
0 kg N ha
1
dry matter)
Lysine (g kg 1
1
crude protein)
100 kg N ha 1
200 kg N ha 1
1991 1992 1993
6.50 ab 6.55 a 6.55 a
6.45 abc 6.15 bc 6.30 abc
6.10 c 6.20 abc 6.10 c
35.20 a 34.32 a 29.15 c
31.92 b 29.36 c 29.56 c
29.95 c 24.32 d 23.55 d
Mean
6.53 a
6.30 b
6.13 b
32.89 a
30.28 b
25.94 c
LSDb (fertilization, P < 0:05) LSDb (year fertilization; P < 0:05)
0.21
0.82
0.36
1.42
a Values of lysine expressed on the same basis (dry matter or crude protein) followed by the different letters are significantly different at the 95% confidence level. b Least significant difference.
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ranged from 2 to 33% for valine and 0 to 14% for lysine. The treatment with 200 kg N ha 1 had a statistically signi®cant effect upon the contents of methionine, valine and lysine in proteins. A signi®cant reduction P < 0:05 was determined both relative to the control and to the treatment with 100 kg N ha 1 in all trial years. The relative decrease, compared to the control, ranged from 37 to 52% for methionine, 14 to 45% for valine, and 13 to 27% for lysine. The effect of the interaction between the nitrogen fertilizer rate and climatic conditions in the trial years on the content of these amino acids in chicory proteins was also signi®cant P < 0:05. Regardless of the in¯uence of climatic conditions, there was a positive signi®cant correlation between the relative decrease of the protein methionine content and the applied nitrogen fertilizer rate (r 0:919, P < 0:01). Fluctuation in the content of other essential amino acids, expressed on the crude protein basis, did not show any regularity with respect to nitrogen fertilizer. 4. Discussion The obtained results show that the content of total nitrogen in chicory dry matter is within the limits of that reported in the literature for head lettuce (Lactuca sativa capitata) (Bergmann and Neubert, 1976), lettuce (Lactuca sativa) (Fontes et al., 1997), and head cabbage (Brassica oleracea capitata) (Krug, 1986). Total nitrogen in chicory dry matter was increased signi®cantly by the higher rate of nitrogen fertilizer in the 2 trial years. Those 2 years were characterized by higher precipitation during the growing season, which might have in¯uenced the overall ion mobility and more intensive leaching of NO3-N from the soil, particularly at its higher concentrations (treatment with 200 kg N ha 1), but also lower nitri®cation and thereby a higher uptake of NH4 -N (Haynes, 1986). Since the interaction between NH4 -N and NO3-N in the soil is very complex (Santamaria et al., 1997b) and NH4 -N presence in soil solution may inhibit the uptake of NO3 -N (Haynes and Goh, 1978), the increase of non-nitrate nitrogen in chicory might be a consequence of a rather high uptake of ammonium nitrogen, which was probably accumulated in the amide form. Marschner (1995) maintains that NH4 inhibits the uptake of NO3 if NH4NO3 fertilizers are applied, as it was the case in our research. This might be a consequence of NH4 deprotonation and easier passage through the membrane (Mengel and Kirkby, 1987). The statistically signi®cant increase of total nitrogen in chicory resulting from higher nitrogen rates is in accordance with similar data for endive reported by Santamaria et al. (1997b). As regards the essential amino acid composition, the data reveal that the plant response to the application of nitrogen fertilizer was manifested differently in the contents of particular essential amino acids in chicory dry matter. A signi®cant decrease was determined for methionine as well as for valine and lysine. Changes of the contents of other essential amino acids, with a tendency to decrease, showed no regularity with regard to the nitrogen fertilizer applied. Similar observations were also reported for other leafy vegetables (MuÈller and Hippe, 1987). Further, the interaction of nitrogen fertilizer rate and climatic conditions was signi®cant P < 0:05 relative to the ¯uctuation of essential amino acid levels in chicory
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dry matter. This is in agreement with the literature data indicating that the interaction between nitrogen availability and soil water is of utmost importance for protein formation in open cropping (Marschner, 1995). However, regardless of the effect of different climatic conditions in the trial years, methionine content was predominantly affected by the increase of the nitrogen rate; the correlation being highly signi®cant. Comparison between trial years for other essential amino acids shows that the smallest changes in the fertilized treatments were recorded in the year with continuing high precipitation during the growing season. It follows from the data obtained that not essential or non-essential amino acids (CÂusticÂ, 1996), but rather amide nitrogen (Whitehead, 1995) contributed to the increase of total nitrogen in chicory dry matter under the in¯uence of nitrogen fertilizer application. Accumulation of inorganic nitrogen, notably NH3, in leafy vegetables in amide form has been reported by a number of authors (Finck, 1982; Yagodin, 1984; Mengel and Kirkby, 1987). The relation between the contents of particular essential amino acids in proteins was similar to their interrelationship in dry matter in the fertilized treatments. However, the relative decrease of certain amino acids in proteins of fertilized chicory, as compared to the control, was substantially higher than the relative decrease of these amino acids expressed on the dry matter basis. This held particularly for the application of the higher nitrogen rate, and was associated with the increase of total nitrogen, namely crude proteins in fertilized treatments. The correlation between the content of all essential amino acids, expressed on the protein basis, and the content of total nitrogen in chicory dry matter was negative and highly signi®cant (Table 6). This observation is consistent with literature data for some other leafy vegetables (Eppendorfer and Bille Sùren, 1996) and indicates depression of the nutritive quality of chicory proteins in fertilized treatments. The effect of nitrogen fertilizer was most pronounced in the relative decrease of methionine in chicory proteins. This may be due to the depression of methionine biosynthesis or to its increased involvement, as the methyl-group donor, in the methylation processes over the course of plant development in the conditions of the higher nitrogen rate application. The results are in agreement with other investigations that also proved high susceptibility of methionine in leafy vegetables connected with the increased Table 6 Correlation coefficients between essential amino acids in proteins and total nitrogen in chicory dry matter r Methionine Valine Lysine Leucine Isoleucine Phenylalanine Threonine **
r 0:01 n 2 0:750. r 0:001 n 2 0:875.
***
0.853** 0.864** 0.974*** 0.831** 0.882*** 0.783** 0.813**
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nitrogen application. Schuphan (1961) reported a signi®cant decrease of methionine content in spinach proteins and so did Eppendorfer and Bille Sùren (1996) for kale proteins and cauli¯ower. 5. Conclusion The obtained results allow the conclusion that the application of nitrogen fertilizer in the production of red head chicory leads to depression of certain essential amino acids, both in dry matter and proteins, at the same time increasing the total nitrogen content. Methionine, valine and lysine levels decrease signi®cantly, particularly with the application of 200 kg N ha 1, where the interaction of the nitrogen rate and weather conditions is signi®cant. It follows from the foregoing that, in the production of red head chicory on hortisol, the nitrogen fertilizer rate supposed to ensure a relatively good nutritive quality of the proteins should not exceed 100 kg N ha 1 in the conditions of optimal humidity. References AOAC, 1975. Methods of Analysis of the Association of Official Analytical Chemists, Washington. Bergmann, W., Neubert, P., 1976. Pflanzendiagnose und Pflanzenanalyse. VEB Gustav Fischer Verlag Jena. CÂusticÂ, M., 1996. Influence of nitrogen fertilization upon the amino acids composition in head chicory. Dissertation, Zagreb. Davies, D.B., Sylvester-Bradley, R., 1995. The contribution of fertiliser nitrogen to leachable nitrogen in the UK: a review. ADAS Soil and Water Research Centre, Cambridge, UK. Elia, A., Santamaria, P., Serio, F., 1998. Nitrogen nutrition, yield and quality of spinach. J. Sci. Food Agric. 76, 341±346. Eppendorfer, W., Bille Sùren, W., 1996. Free and total amino acid composition of edible parts of beans, kale, spinach, cauliflower and potatoes as influenced by nitrogen fertilisation and phosphorus and potassium deficiency. J. Sci. Food Agric. 68, 449±458. Finck, A., 1982. Fertilizers and Fertilization. Introduction and Practical Guide to Crop Fertilization. Weinhei/ Deerfield Beach, Florida/Basel. Fontes, P.C.R., Pereira, P.R.G., Conde, R.M., 1997. Critical chlorophyll, total nitrogen, and nitrate-nitrogen in leaves associated to maximum lettuce yield. J. Plant Nutr. 20, 1061±1068. Haynes, R.J., 1986. Mineral Nitrogen in the Plant±Soil System. Academic Press, Orlando, FL. Haynes, R.J., Goh, K.M., 1978. Ammonium and nitrate nutrition of plants. Biol. Rev. 53, 465±510. Krishnamurti, C.R., Heindze, A.M., Galzy, G., 1984. Application of reversed-phase high-performance liquid chromatography using pre-column derivatisation with o-phthaldialdehyde for the quantitative analysis of amino acids in adult and fetal sheep plasma, animal feeds and tissues. J. Chrom. 315, 321±331. Krug, H., 1986. GemuÈseproduktion, Verlag Paul Parley, Berlin und Hamburg. Marschner, H., 1995. Mineral Nutrition of Higher Plants, 2nd Edition. Academic Press, London. Maynard, D.N., Barker, A.V., Minotti, P.L., Peck, N.H., 1976. Nitrate accumulation in vegetables. Adv. Agron. 28, 71±118. Mengel, K., Kirkby, E.A., 1987. Principles of Plant Nutrition. International Potash Institute, Bern. MuÈller, K., Hippe, J., 1987. Influence of differences in nutrition on important quality characteristics of some agricultural crops. Plant Soil 100, 35±45. Newbould, P., 1989. The use of nitrogen fertiliser in agriculture. Where do we go practically and ecologically. Plant Soil 115, 297±311. Santamaria, P., Elia, A., 1997. Producing nitrate-free endive heads: effect of nitrogen form on growth, yield and ion composition of endive. J. Am. Soc. Hort. Sci. 122, 140±145.
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Santamaria, P., Elia, A., Gonnella, M., Serio, F., 1997a. Ways of reducing nitrate content in hydroponically grown leafy vegetables. In: Proceedings of the Ninth International Congress on Soilless Culture, pp. 437±451. Santamaria, P., Elia, A., Gonella, M., 1997b. Changes in nitrate accumulation and growth of endive plants during light period as affected by nitrogen level and form. J. Plant Nutr. 20, 1255±1266. SAS, 1989. SAS/STAT User's Guide, Ver. 6, Vols. 1 and 2, 4th Edition. SAS Institute, Inc., Cary, NC. Schuphan, W., 1961. Methioningehalt und EiweisqualitaÈt von Blattpflanzen in AbhaÈngigkeit von der StickstoffduÈngung. Qual. Plant. Mat. Veg. 8, 261±283. Steel, R.G.D., Torrie, J.H., 1980. Principles and Procedures of Statistics. McGraw-Hill, New York. Walker, R., 1990. Nitrates, nitrites and N-nitroso compounds: a review of the occurrence in food and diet and the toxicological implications. Food Addit. Contam. 6, 717±768. Whitehead, D.C., 1995. Grassland Nitrogen. CAB International, Oxon, UK. Yagodin, B.A., 1984. Agricultural Chemistry. Mir, Moscow.