Effects of nickel concentration in the nutrient solution
on the nitrogen assimilation and growth of tomato
seedlings in hydroponic culture supplied with urea
or nitrate as the sole nitrogen source
Xue Wen Tan, Hideo Ikeda
*, Masayuki Oda
Laboratory of Vegetable Crops, College of Agriculture, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan
Accepted 13 August 1999
Abstract
Both the bene®cial and the adverse effects of nickel supplement on the N assimilation and growth of tomato plants were evaluated while either urea or nitrate was applied as the sole N source in the nutrient solution.
The nickel concentration in the plant was related to that in the solution. Urea toxicity to the plants was reduced by the nickel supplement at 0.01 mg lÿ1
, and no symptom of urea toxicity was observed in the plants when supplemented nickel was at 0.1 or 1 mg lÿ1. Both the plant growth and
chlorophyll concentration in leaves of the urea-fed plants increased when nickel concentration in the solution was up to 0.1 mg lÿ1, but decreased by nickel at 1 mg lÿ1. The maximum growth of the
urea-fed plants promoted by nickel supplement was still less than 80% of the nitrate-fed plants. Nickel supplement did not affect the growth of the nitrate-fed plants.
The concentrations of leaf total-N in the urea-fed plants was enhanced by the nickel supplement, but was still lower than those in the nitrate-fed plants. At 0.1 mg lÿ1
nickel supplement, the concentrations of leaf urea-N and NH4-N in the urea-fed plants were about one-sixth and four times,
respectively, of those without nickel supplement. Urea assimilation increased with the nickel supplement up to 0.1 mg lÿ1. No effect of nickel supplement on the N assimilation in the nitrate-fed
plants was found.
Nickel supplement up to 0.1 mg lÿ1
reduced urea toxicity and enhanced chlorophyll concentration, plant growth, and urea hydrolysis. The symptom of nickel toxicity and the
Scientia Horticulturae 84 (2000) 265±273
*
Corresponding author. Tel:81-722-54-9421; fax:81-722-54-9918.
E-mail address: ikeda@plant.osakafu-u.ac.jp (H. Ikeda).
depression of plant growth were only observed with the nickel supplement up to 1 mg lÿ1
.#2000 Elsevier Science B.V. All rights reserved.
Keywords: Tomato; Nickel; Urea; Growth; Urea toxicity; N assimilation
1. Introduction
Urea is an important nitrogen (N) fertilizer in today's agriculture and its suitability for ®eld crops has been well evaluated (Vavrina and Obreza, 1993). However, urea in comparison to nitrate is not a desirable hydroponic N source for tomatoes (Kirkby and Mengel, 1967; Ikeda and Tan, 1998), lettuces (Luo et al., 1993), zucchinis (Gerendas and Sattelmacher, 1997), and rices (Gerendas et al., 1998) due to its toxicity. The possible cause of urea toxicity is either by the NH
4 released during urea assimilation (Luo et al., 1993) or by urea itself (Krogmeier et al., 1989). This latter conclusion was supported by a subsequent experiment in which nickel de®cient plants showed lower urease activity and more leaf-tip necrosis than non-de®cient plants (Krogmeier et al., 1991). Because urease is the ®rst enzyme involved in urea assimilation by plant tissues for the hydrolysis of urea (Hogan et al., 1983), one possible way to avoid urea toxicity is to increase the activity of urease.
Nickel is an essential micronutrient for some higher plants (Brown et al., 1987), and the enzyme urease from jack beans (Dixon et al., 1975) and soybean seed (Polacco and Havir, 1979) is known to be a nickel metalloenzyme. Therefore, nickel is considered to be an important element for plants applied with urea as a N source due to its role in the metalloenzyme urease (Gerendas et al., 1998). The possible way to avoid urea toxicity is to use nickel supplement to enhance the urease activity (Bekkari and Pizelle, 1992; Marschner, 1995).
Although the requirement of nickel for the urease activity was discovered more than 20 years ago (Dixon et al., 1975), effects of nickel concentration in the urea solution on the growth have received little attention. In this study, we tested whether the nickel supplement can reduce urea toxicity and investigated effects of nickel concentration in the solution on the N assimilation and growth of tomato plants in hydrophonic culture supplied with urea or nitrate as the sole nitrogen source.
2. Materials and methods
2.1. Plant materials, growth conditions, and treatments
emergence, were transferred to a hydrophonic culture in a greenhouse under natural sunlight during autumn (average air temperature 32/208C day/night). A half strength Hoagland's nutrient solution was used before the onset of treatments.
When the seedlings had grown to the 6±7 leaf stage, eight plants were transplanted into a 15 l polypropylene container. The basic nutrient solution was prepared as follows: (in mM) K2SO4: 2, CaCl22H2O: 1.5, MgSO47H2O: 1,
NaH2PO42H2O: 2/3, and micronutrients. Plants were treated with two N sources:
urea and nitrate as NaNO3 at 168 mg N l
ÿ1
, and four concentrations of nickel as NiSO46H2O: 0, 0.01, 0.1, and 1 mg l
ÿ1
. Treatments were arranged in a randomized block design with three replicates. All solutions were prepared with deionized water, aerated using an aquarium air stone, and were renewed every 7 days. Solution pH was daily checked and adjusted to 6.0 with 1 N NaOH or HCl.
2.2. Investigations, samplings and chemical analyses
Before harvest, the concentrations of leaf chlorophyll were measured with a hand-held chlorophyll meter (SPAD-502, Minolta), and visual leaf symptoms of urea toxicity were assessed with index from 0 (none) to 3 (severe). Plants were harvested 4 weeks after treatment and were divided into leaves, stems, and roots. These plant materials were then dried immediately in a forced-air oven at 608C to a constant weight, weighed, and were ground to a ®ne powder in a Wiley mill to pass through a 20-mesh sieve. The concentrations of total-N and nickel in the leaf samples were determined by a modi®ed Kjeldahl method and ICP, respectively. To determine the concentrations of urea-N, NH4-N, and NO3-N, the samples were
extracted with hot water. The concentrations of urea-N were determined using the method of Cline and Fink (1956), and the concentrations of NH4-N and NO3-N
were determined using ion exchange chromatography (Dionex DX-AQ).
A statistical analysis was made using analysis of variance, and the means were separated by Duncan's multiple range test (DMRT) at the 5% level.
3. Results
Leaf nickel concentrations in the urea-fed plants increased signi®cantly with the increase of the nickel concentration in the solution from 0.01 to 1 mg lÿ1
: 7.5 times from 0.01 to 0.1 mg lÿ1
and 13.9 times from 0.1 to 1 mg lÿ1
(Fig. 1). Leaf nickel concentrations in the urea- or nitrate-fed plants without nickel supplement were relatively low. N source did not signi®cantly affect leaf nickel concentration. The symptoms of urea toxicity were severe in the urea-fed plants without nickel supplement, and the symptoms were reduced in the plants with nickel supplement at 0.01 mg lÿ1
(Fig. 2). No symptom of urea toxicity was observed in the plants supplemented with nickel at 0.1 or 1 mg lÿ1
.
Fig. 1. Effects of nickel concentration in the nutrient solution on the leaf nickel concentration in the tomato plants applied with urea or nitrate. Leaf samples were obtained 4 weeks after treatment. Bars having different letters are signi®cantly different at the 5% level by DMRT.
The dry matter increments of the shoots or roots of the urea-fed plants increased signi®cantly with the increase of the nickel supplement from 0 to 0.1 mg lÿ1
, but decreased sharply with nickel at 1 mg lÿ1
(Table 1). The highest dry matter increment in plants fed with urea at 0.1 mg lÿ1
nickel supplement was 78% of the nitrate-fed plants without nickel supplement. The dry matter increments of the shoots or roots of the nitrate-fed plants were not affected by nickel supplement.
With urea nutrition, leaf chlorophyll concentrations were low in the plants without nickel supplement and in the plants supplemented with nickel at 1 mg lÿ1
(Fig. 3). Leaf chlorophyll concentrations in the nitrate-fed plants were not signi®cantly affected by the nickel supplement.
The lowest concentrations of leaf total-N were detected in the urea-fed plants without nickel supplement (Table 2). The concentrations of leaf urea-N and its percentages in the total-N decreased greatly with the increase of the nickel supplement from 0 to 0.1 mg lÿ1
, but no further reduction at 1 mg lÿ1
. Although the concentrations of leaf NO3-N in the urea-fed plants were not affected by the
nickel supplement, the concentrations of leaf NH4-N increased sharply with the
increase of the nickel supplement. The concentrations of leaf urea-N and NH4-N
in the urea-fed plants with nickel supplement at 0.1 mg lÿ1
were about one-sixth and four times of those without nickel supplement, respectively. Urea assimilation increased as the nickel concentration in the solution increased from 0 to 0.1 mg lÿ1
, but no further increase at 1 mg lÿ1
. Higher concentrations of total-N were detected in the nitrate-fed plants than in urea-fed plants. No effects of nickel supplement on the concentrations of total-N, NH4-N, NO3-N, and the assimilation
of nitrate in the nitrate-fed plants were found.
Table 1
Effects of nickel concentration in the nutrient solution on the dry matter increments of tomato plants fed with urea or nitrate for 4 weeksa
Nitrogen
Urea 0 2.29d 0.70b 2.99d 43
0.01 3.09c 0.91a 4.00c 58
0.10 4.34b 1.07a 5.41b 78
1.00 2.52d 0.69b 3.21d 46
Nitrate 0 5.98a 0.94a 6.92a 100
0.10 6.05a 0.93a 6.98a 101
a
Means in each column followed by different letters are signi®cantly different at the 5% level by DMRT. DW increment: DW (4 weeks after treatment)ÿDW (before treatment).
b
The total dry matter increments of the nitrate-fed plants without nickel supplement is represented as 100.
4. Discussion
We have shown that nickel in the nutrient solution was absorbed by the plants as the leaf nickel concentration was signi®cantly increased by the increase of the nickel concentration supplemented in the nutrient solution.
Fig. 3. Effects of nickel concentration in the nutrient solution on the leaf chlorophyll concentrations of the tamto plants applied with urea or nitrate. Values were SPAD readings from chlorophyll meter (SPAD-502, Minolta) 4 weeks after treatment. Bars having different letters are signi®cantly different at the 5% level by DMRT.
Table 2
Effects of nickel concentration in the nutrient solution on the concentration and assimilation of N in the leaves of tomato plants fed with urea or nitrate for 4 weeksa
Nitrogen source
Nickel concentration
Total-N (mg gÿ1
)
Urea-N (mg gÿ1
) NH
4-N
(mg gÿ1
) NOÿ
3-N
(mg gÿ1
)
Urea-N/ total-N (%)
Assimilated (%)b
Urea 0 35.81c 13.14a 0.91c 0.50b 36.7a 63.3c
0.01 39.74b 5.68b 1.83b 0.55b 14.3b 85.7b
0.10 41.14b 2.22c 3.55a 0.49b 5.4c 94.6a
1.00 40.86b 2.35c 3.54a 0.49b 5.8c 94.2a
Nitrate 0 55.04a 1.09c 9.64a 82.5b
0.10 54.90a 1.10c 9.46a 82.8b
aMeans in each column followed by different letters are signi®cantly different at the 5% level by
DMRT. Data were obtained 4 weeks after treatment.
b
The concentrations of leaf urea-N in the urea-fed plants without nickel supplement were relatively high and were decreased signi®cantly with the nickel supplement. The concentrations of leaf urea-N in the plants supplemented with nickel at 0.1 mg lÿ1
were only about one-sixth in the plants without nickel supplement, while the concentrations of leaf NH4-N in the former plants were
about four times of the latter plants. The changes of the concentrations of leaf urea-N and NH4-N in the urea-fed plants indicate that a very rapid initial
hydrolysis of urea is stimulated by the nickel absorbed from the nutrient solution. This result is in agreement with the ®ndings by Nicouland and Bloom (1998) with tomatoes. The rapid hydrolysis of urea may be caused by higher activity of urease activated by the addition of nickel (Eskew et al., 1984; Brown et al., 1987).
Nickel supplement to the urea solution reduced the symptom of urea toxicity. The toxicity is caused by urea rather than NH
4, the urea assimilation product, for the following reasons: (1) the concentration of leaf urea-N decreased with the nickel supplement, and the lower urea-N concentration was accompanied by less toxicity symptom; (2) the symptom of toxicity reported here was different from that of NH
4 toxicity. In this study, the toxicity symptom was the brown color around the leaf margin, while partial leaf yellowing, mottled chlorosis, and curing the symptoms of NH
4 toxicity in our previous experiment with tomato; and (3) As the concentration of leaf NH
4 increased greatly with the nickel supplement in urea-fed plants, while the urea toxicity symptom was reduced; urea toxicity is unlikely caused by NH
4 in the leaf.
Nickel supplement promoted the growth of urea-fed plants, and the growth promotion is related to the improvement of urea assimilation. In this study, both the urea assimilation and the chlorophyll concentration in the urea-fed plants increased when nickel was supplemented. The increase of the chlorophyll concentration demonstrates strongly the improvement of urea assimilation by the nickel supplement because chlorophyll is one of the important N-containing compounds (Barker, 1989). Minotti et al. (1994) demonstrated that the N status in the plant can be assessed by the measurement of chlorophyll. The growth promotion is also related to the absorption of urea. The concentrations of leaf total-N in the urea-fed plants with nickel supplement were higher than those without nickel supplement, indicating that the absorption of urea is increased by the nickel supplement. A rapid growth of the urea-fed plants was obtained with nickel supplement at 0.1 mg lÿ1
, while is about 80% of the nitrate-fed plants. We think that the growth difference between urea- and nitrate-fed plants is caused by the difference of N absorption. Although the concentrations of leaf total-N in the urea-fed plants increased with the nickel supplement, they were still lower than those of the nitrate-fed plants.
Although nickel supplement to the urea solution promoted plant growth, nickel at high concentration depressed plant growth. The growth depressed by the high nickel concentration may be caused by nickel toxicity because many small black
spots on the stems and brown roots were observed in the plants supplemented with nickel at 1 mg lÿ1
. The symptoms are caused by nickel itself, rather than by urea or NH
4: (1) these symptoms differ from those caused by urea or NH
4 toxicity and (2) the concentration of leaf urea-N or NH4-N in the plants
supplemented with nickel at 0.1 mg lÿ1
was similar to that supplemented with nickel at 1 mg lÿ1
, but these plants had no nickel toxicity symptoms.
The mechanisms of nickel toxicity to the plant are still unclear. Gabbrielli et al. (1990) reported that root growth and cell division ofSilene italicais sensitive to the nickel concentration in the solution. On the other hand, Yang et al. (1997) found that the tolerance of some species to the nickel concentration depends on the interaction of nickel with organic acids.
Acknowledgements
We thank all the students in our laboratory for their assistance during the experiment. This study was supported by Grants-in-Aid for Scienti®c Research (H. Ikeda: No. 08456023) from the Ministry of Education, Science, Sports, and Culture of Japan.
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