tions but additionally it may originate from the different experimental techniques used.
In order to distinguish between the soil N and root borne N, plant N must be labelled and this is
generally done using
15
N. Reining et al. 1995 used the ‘split-root-technique’ in order to label the
plant N. This method allows the precise quantifi- cation of released N from the root part not in
contact with the labelled soil N. This procedure, however, does not allow the quantitative determi-
nation of labelled N released by the entire root system. Janzen and Bruinsma 1989 labelled their
plants by the uptake of
15
NH
3
by the upper plant parts. Unfortunately the amount of labelled N
taken up is small and therefore it is difficult to quantify the release of labelled N by the roots
over longer periods of time. Since the labelling methods used to date all have their shortcomings
Palta et al., 1991, we have developed a new technique for labelling entire plants with
15
N which is particularly suitable for measuring N
losses from tillering until maturation. The princi- ple of our method is the labelling of seedlings in
15
N nutrient solution prior to planting into pots filled with soil.
The objective of our investigation was to quan- tify the labelled N released from intact wheat
roots into the soil as well as other losses mainly into the atmosphere at distinct growth stages of
the plants: tillering, ear emergence, beginning of grain filling, and maturation.
2. Materials and method
The experiments were carried out in 1997 and 1998. In the 1st year the plants were cultivated in
a semi-open glasshouse up to ear emergence and were then transferred into a growth chamber. In
the 2nd year cultivation was in a growth chamber throughout the growth period. The light intensity
was 180 mmol E m
− 2
s
− 1
in a 16-h photoperiod with a 24°C day temperature and a 14°C night
temperature. The relative air humidity ranged from 45 to 55. Spring wheat seeds were soaked
in a CaSO
4
0.5-M solution for 24 h and then allowed to germinate on filter paper over a 4-day
period. The seedlings were then cultivated in a
15
N nutrient solution with the following composi- tion: 4 mM K
2
SO
4
, 2 mM MgSO
4
, 0.3 mM NaH
2
PO
4
, 2 mM NH
4
NO
3
, 4 mM CaCl
2
, 2 mM H
3
BO
3
, 0.1 mM CuSO
4
, 0.01 mM Na
2
MoO
4
, 0.2 m
M MnSO
4
, 0.1 mM ZnSO
4
, 100 mM Fe as FeEDTA. The NH
4
NO
3
was double labelled at 98 atom excess. During the first days plants re-
ceived 14 of the full concentration of the nutrient solution, then 12, and 8 days later the full con-
centration. The seedlings were cultivated in the nutrient solution for 3 weeks, this time being
required to attain a sufficient amount of
15
N in the plants. The plants were then removed from
the nutrient solution and their roots were thor- oughly rinsed with distilled water in order to
remove adhering
15
N. After this the young plants were transplanted into soil in cultivation pots.
Plants were held in the pot with their basal root section at the level of the edge of the pot and then
dry soil was carefully added so that all roots were in close contact with the soil. Each pot held three
plants and received 900 g soil fertilised with 140 mg K and 56 mg P as K
2
HPO
4
, and 160 mg N as NH
4
NO
3
. The soil was a silty loam derived from loess whose characteristics are shown in Table 1.
Table 1 Characteristics of the soil used in the two experiments
Silt Clay
Sand K CAL
CaCl
2
extraction Tot N
P CAL
a
pH mg kg
− 1
g kg
− 1
g kg
− 1
mg kg
− 1
g kg
− 1
mg kg
− 1
g kg
− 1
CaCl
2
Norg NH
4
NO
3
5.8 210
730 60
4.65 2.65
7.80 1.63
1.3 2.62
a
CAL, Ca-acetate-lactate method.
Since the plants had to grow for 3 weeks in nutrient solution and because of the high cost of
15
N it was decided not to change the nutrient solution and thus discard the
15
N, but to add the nitrogen to the nutrient solution according to its
uptake by the plants. It is known that nitrogen uptake by plants has
an impact on the pH of the nutrient solution, ammonium uptake depressing the solution pH
and nitrate uptake raising it Mengel et al., 1983. According to this finding preferential utilisation
of ammonium in the nutrient solution is associ- ated with a pH decrease and likewise that of
nitrate is indicated by an increase in pH in the nutrient solution. In order to confirm this rela-
tionship for our experimental set-up a preliminary experiment was carried out with 104 spring wheat
seedlings which were set in a 20-l plastic container and cultivated in the nutrient solution described
above with the exception that the nitrogen was not labelled with
15
N. Cultivation lasted 22 days and during this period, pH of the nutrient solu-
tion was measured twice a day and the pH was adjusted to the initial pH level by adding KOH or
H
2
SO
4
0.1 M, respectively. At the same time samples were taken from the nutrient solution to
determine the ammonium and nitrate concentra- tions. The volume of nutrient solution was filled
up twice a day to 20 l by adding distilled water.
The total N content in the plants roots + shoots
of the
preliminary experiment
was analysed by the Kjeldahl method and the NH
4 +
and NO
3 −
concentrations in nutrient solution were also analysed by the Kjeldahl method using MgO
for distillation and Devarda alloy for reduction of nitrate.
The
15
N release into the soil in the
15
N experi- ments was analysed at tillering, ear emergence,
beginning of grain filling and full maturation. At each development stage plants of two pots were
harvested and analysed. In one pot shoots and roots were analysed separately for total N and
15
N after the soil was washed carefully from the roots. In the other pot roots + soil were taken
together, ground and analysed for
15
N enrichment and total N.
15
N of this pot + the
15
N in the shoot gave the total amount of
15
N in the soil-plant system. The
15
N amounts released into the soil were calculated according the following equation:
15
N released in the soil = [
15
Nsoil + roots +
15
N shoots] −
[
15
N shoots +
15
N roots] 1
Losses unaccounted for which could not be measured directly were calculated according to
the second equation: Unaccounted for losses =
{
15
Nsoil + roots +
15
N shoots} at tillering −
{
15
Nsoil + roots +
15
N shoots} at different growth stages
2 These losses include gaseous N losses and losses
of plant material. According to Handley and Raven 1992 en-
zymes discriminate
15
N relative to
14
N which may lead to difference in d
15
N of maximum 12‰. This means that in an enzymic reaction the quantity of
a
15
N metabolite converted is 1 lower than the same amount of a
14
N analogue metabolite. Assuming that
15
N nitrate assimilation and fur- ther conversions of the
15
N metabolite would comprise the reactions nitrate reduction, nitrite
reduction, ammonium assimilation and polypep- tide synthesis four reactions, the discrimination
would be 4 and the data found would be 4 lower than the real amount. In the case of ammo-
nium assimilation the discrimination would be
2 since here the steps of nitrate and nitrite reductions are not involved. This small difference
has no major impact on our results and the conclusions drawn.
The washing of soil from roots was done very carefully in order to keep to a minimum the losses
of plant material. This process was carried out using a very slight water flow, over a sieve system
containing two sieves the first of 1 mm and the second of 0.15 mm. The separated root pieces,
were collected in the two sieves and put together with the other roots. To quantify the possible
losses of labelled N through root washing a finer sieve of 0.06 mm was tested for some pots for
plants harvested at the beginning of grain filling and full maturation. This test showed that using
the 0.06-mm sieve only 0.026 and 0.084 mg
15
N pot
− 1
were recovered at the beginning of grain
Fig. 1. Changes in the nutrient solution pH due to NH
4 +
and NO
3 −
uptake by wheat seedlings. Dotted lines show the pH correction by the addition of H
2
SO
4
or KOH, respectively.
filling and full maturation, respectively. These small quantities, which may be accounted for by
losses of root hairs, amount merely to 0.1 and 0.25 of the total
15
N in the plant + soil at the beginning of grain filling and full maturation,
respectively. In the mixed sample soil + roots second pot
60 mg dry matter was needed for
15
N analysis. For this purpose the total amount of soil + roots
was dried and ground together. The mixed sample was than treated twice with a sample separator
Retsch to obtain a representative subsample of
10 g for
15
N analysis. To prove whether the sample separation did influence the analytical re-
sults, several subsamples taken from one soil + root sample were analyzed for
15
N enrichment. The
15
N concentrations of these subsamples did not differ statistically from one another.
It was not possible to measure
15
N in the plants at the date of transplantation because of the very
high
15
N enrichment values over 80, and be- cause with the equipment used Vario EL and
N01-6 only a limited concentration of
15
N in plant dry matter can be measured. We therefore
used the
15
N in the plants + soil at tillering as a control at which stage the
15
N in the plant had been diluted to 66.
The total N and
15
N excess analysis were car- ried out using a Vario EL elementary analyser,
connected with an N01-6 emission spectrometer Fischer, Germany at the Institute of Plant Nu-
trition and Rhizosphere Research in ZALF, Mu¨ncheberg.
In the 1st year the experiment included eight, and the 2nd year, six biological replications.
Statistical elaboration of the data was done using the t-test according to Ko¨hler et al. 1996 for
measuring statistical differences between pairs and ANOVA, and t-test for the statistical differences
between growth stages.
3. Results
