Philippine Journal of Crop Science (PJCS) August 2005, 30(2): 21-28
Copyright 2005, Crop Science Society of the Philippines
Released 19 June 2005
ISOTOPE-LABELLING OF PLANT MATERIALS TO STUDY
CARBON, NITROGEN AND SULFUR DYNAMICS IN SOILS
PEARL BASILIO-SANCHEZ1, GRAEME BLAIR2, RAY TILL2 & MICHAEL FAINT2
1
Assistant Professor, University of the Philippines Los Baños, College, Laguna 4031 Philippines; 2 Agronomy and Soil Science, University of New England,
Armidale, NSW 2351 Australia
A labelling technique to produce large quantities of plant materials multi-labelled with 14C, 15N, 35S and 13C, 14C, 15N was developed
to study the fate of residue-derived C, N and S following incorporation into the soil. Flemingia (Flemingia macrophylla), medic
(Medicago truncatula) and rice plants were grown in pots and labelled with 15N and 35S by adding 15N and 35S solutions every 4 days
for 6 weeks. A labelling chamber measuring 2.5 x 1.3 x 1 meters constructed from aluminum and PVC frames and covered with ethylvinyl alcohol film, was used to label the plant materials with 14CO2 and 13CO2. Labelled CO2 was generated inside the chamber by the
reaction of Na214CO3 or Na213CO3 with lactic acid and circulated by a commercial air-conditioning unit set in a recycling mode. The
level of CO2 within the chamber was monitored using an infrared gas analyzer (ADC Type 225 MK3 CO2 Analyzer). The 14C-, 15N-,
35
S-labelled plant materials were used in a glasshouse experiment to monitor the decomposition process and trace the fate of residuederived C, N and S in the presence of a millet crop. The labelled residues of medic hay and Flemingia leaves were dried, cut into 2-3
cm length and incorporated into a sandy loam soil at a rate of 3 tons/ha. Results showed that medic hay had a faster decomposition
rate than Flemingia leaves, with almost half of its 14C disappearing after 41 days. The addition of medic hay resulted in significantly
higher 15N and 35S recoveries in the first millet crop and lower 15N and 35S recoveries in the soil. Flemingia leaves released smaller
proportions of 15N and 35S that resulted in higher 15N and 35S recovery in the soil and in the second crop of millet. The isotopic
enrichments attained were sufficient to show major differences between medic hay and Flemingia leaves as residues with respect to
their usefulness as nutrient source and to the sequestration of carbon in the soil. A plant residue with a rapid decomposition rate like
medic can provide an immediate source of nutrients particularly for short-duration crops; a residue that decomposes slowly like Flemingia
results in a gradual release of nutrients and hence can have longer impact on crop growth.
13
C, 14C, 15N, 35S, crop residue, decomposition, Flemingia, isotope-labelling, Medicago, medic hay, millet, nutrient release, nutrient
transformation, rice
INTRODUCTION
Isotope-labelled plant materials have been widely used to
study decomposition rates, nutrient release patterns and
transformation of plant-derived nutrients in soils. Early
decomposition studies made use of 14C-labelled plant materials
produced by growing plants in an atmosphere continuously
enriched with 14CO2 (Jenkinson 1965, Shields & Paul 1973,
Jenkinson & Ayanaba 1977, Ladd et al 1981). Numerous
designs of carbon isotope-labelling chambers have been
published, ranging from simple enclosures consisting of a
polyethylene tent that can be punctured and sealed (Dahlman &
Kucera 1968) to rigid chambers with sophisticated control
equipment (Wagner et al 1995) where plants can be exposed to
a single large dose of 14CO2 or to repeated 14C pulses depending
on the researcher’s needs and resources. Due to health and
environmental hazards associated with the use of radioactive
materials, along with the increasing availability of equipment
for the rapid analysis of 13C, interest in the use the use of the
stable carbon isotope 13C has increased (Berg et al 1991,
Thompson 1996).
Nutrient cycling studies have also used 15N- and 35Slabelled plant materials (Till et al 1982, Konboon et al 2000),
and since carbon is closely linked to nutrient cycling, duallabelled plant materials (14C/15N, 13C/15N, 13C/35S) have been
used (Broadbent & Nakashima 1974, Amato & Ladd 1980,
Amato et al 1984, Aita et al 1997, Hopkins et al 1997, Bottner et
al 1998, Konboon et al 2000).
This research was undertaken to develop a technique to
produce triple-labelled (14C, 15N, 35S and 13C, 14C, 15N) Flemingia
(Flemingia macrophylla), medic (Medicago truncatula) and
rice (Oryza sativa) plant materials. Flemingia is a droughtresistant shrub legume well-adapted to low-fertility, acid soils of
the humid tropics (Andersson et al 2003).
The efficiency of the multiple-labelling technique in
incorporating both stable and radioactive carbon isotopes into
the plant tissue was also explored. A glasshouse experiment was
later conducted to trace the fate of carbon, nitrogen and sulfur
released from the decomposition of labelled Flemingia leaves
and medic hay.
The comparative use of labelled Flemingia and medic,
MATERIALS & METHODS
Production of labelled plant materials
Growth and maintenance of plants
Flemingia, medic and rice were grown in 1:1
vermiculite:sand mixture in plastic-lined pots to prevent isotopic
contamination of the surroundings. Once the seedlings were
established, each pot was thinned to 2 Flemingia, 10 medic and
6 rice plants. The pots were watered to field capacity by weight.
Nutrient solution (Aquasol supplemented with MgSO4) was
applied once every fortnight, then increased to weekly as plant
growth increased. Pesticides were applied when necessary.
CO2
Air
con
IRGA
Figure 1.
Schematic diagram of the labelling chamber used
which have different decomposition rates (Konboon 1997),
provides an opportunity to measure turnover rates of crop
residues in farming systems where prunings of leguminous
shrubs/trees such as Flemingia and legume crops such as medic
are used as green manure. Labelled rice straw is particularly
useful in studying decomposition in agricultural systems where
the straw is left on the soil surface after harvest or incorporated
into the soil.
22
Flemingia plants were grown to about 50 cm, then heavily
defoliated, and the medic and rice plants severely pruned before
labelling to ensure that the newly formed plant tissue would be
highly enriched and uniformly labelled.
Labelling chamber
The chamber (Figure 1) was 2.5 meters long, 1.3 meters
wide and 1 meter high, large enough to produce large quantities
Isotope-Labelling Of Plant Materials
of plant materials needed in the incubation studies. The frame
was made from aluminum and PVC pipes and the sides from
clear, gas-proof ethyl-vinyl alcohol film (Trigon Packaging
System Ltd, Christchurch NZ). The three sides of the chamber
were permanently taped to the floor and sandbags were used to
produce a permanent and gas-tight seal. The front was left
unsealed to allow watering of plants and application of 15N and
35
S solution. The 14CO2 and 13CO2 were generated from the
reaction of labelled Na2CO3 with lactic acid injected through a
thin plastic tube that ran through the side of the labelling
chamber.
A commercial air-conditioning unit was attached on one
side to circulate the air inside the chamber and at the same time
regulate the temperature to about 25°C. During cloudy days and
when additional lighting was required, mercury vapor lamps
located in the glasshouse above the chamber were turned on.
The CO2 concentration inside the chamber was monitored by an
infrared gas analyzer (ADC Type 225 Mk3 CO2 analyzer).
C, 14C and 15N labelling
The technique for producing 13C-, 14C- and 15N-labelled
plant materials was similar to that described earlier except that
Na213CO3 was mixed with the Na214CO3 solution before addition
of lactic acid to liberate 13CO2 and 14CO2 inside the chamber. The
same Flemingia plants grown for the first labelling were used
while new medic and rice plants were established. The plants
were again heavily pruned prior to labelling.
The amount of Na213CO3 (99%) used for each pulse
increased from 0.5 grams in the first week to 1.0 grams in the
second and third weeks then to 1.9 grams for the succeeding
weeks. It was reduced to 1.0 gram when only the rice plants
were left to mature. A constant pulse of 2.5MBq 14C was
administered throughout the labelling period. Since the different
plant species were not harvested at the same time, they were
exposed to varying amounts of CO2 released: 21.6 g Na213CO3
and 32.5 MBq 14C for medic, 33.0 g Na213CO3 and 47.5 MBq 14C
for Flemingia, and 37.0 g Na213CO3 and 57.5 MBq 14C for rice.
14
Harvesting
C, 15N and 35S labelling
The pulses of labelled CO2 were administered between
9:00-10:00 AM when photosynthetic activity was expected to
be high. During cloudy days, the mercury vapor lights above the
chamber were turned on to maintain a daylength of 12 hours.
The CO2 concentration inside the chamber was allowed to drop
from 350 to 300 ppm before the 14CO2 pulse was introduced.
When the CO2 concentration declined to approximately 180
ppm and became steady, 12CO2 was introduced into the chamber
from a gas cylinder to return the concentration to 350 ppm.
Sequential 12CO2 pulsing continued for the rest of the day or
until 6:00 PM when artificial lighting was used, to maximize
uptake of 14CO2. The chamber was closed overnight to contain
any 14CO2 released during respiration and prevent any 14CO2
leaking into the atmosphere. Additional pulses of 12CO2 were
administered the next day before opening the chamber to
expose the plants to natural conditions.
Although 2.5 MBq of labelled C was administered each
time, the frequency of labelling increased from once a week to
four times a week as the plant biomass increased. The plants
were pulse-labelled with 14CO2 15 times during the growing
season, the pots rotated prior to each labelling to ensure
uniformity of labelling among pots.
15
N- and 35S-labelling were accomplished by adding 10
mL of solution containing 0.33mg15N/mL and 93 kBq 35S/mL
every 4 days for 6 weeks. The 15N source was 15NH4Cl added at
the rate of 3.30mg 15N/pot and carrier-free 35S added at a rate of
0.93 MBq 35S/pot. These were added to the surface of the pot
using a syringe.
Pearl Basilio-Sanchez et al
13
The medic and rice plants were cut close to the surface of
the potting medium while the Flemingia leaves were separated
from the stem. The labelled plant materials from all the pots of
the same species were bulked before oven-drying at 50°C.
Medic tops, rice straw and Flemingia leaves were cut into 2-3
cm lengths, mixed to ensure uniformity, then stored for the
glasshouse experiments.
Glasshouse experiment
The soil used was a sandy loam collected from the upper
30 cm of an unfertilized native pasture near Uralla, NSW,
Australia. Bulk soil was passed through a rotor to remove plant
materials, air-dried, sieved to pass a 2-mm sieve and stored at
room temperature prior to use. The soil contained 0.84% total
C, 0.074% total N and had a δ13C –16.74‰.
Polyvinyl chloride (PVC) cylinders (30 cm x 15-cm inner
diameter) were divided into three 9-cm layer; each filled with
2.1 kg soil, and referred to as top, middle and bottom layers. The
labelled plant materials were incorporated in the surface 5 cm of
the top layer at a rate equivalent to 3 tons/ha, one day after the
application of basal macro and micro nutrients. Japanese millet
(Echinochloa crus-galli var. frumentacea) was sown at a rate of
10 seeds/pot 2 days after residue application (DAA) and later
thinned to 6 seedlings/pot.
Three replicates of each residue, including a control
treatment with no residue, were laid out in a randomized
complete block design. Japanese millet was harvested 41 and 71
DAA. Another crop of millet was established and harvested after
a further 37 days (108 DAA). Correspondingly, the soil was
destructively sampled at 41, 71 and 108 DAA.
23
Sample preparation
Plant samples were oven-dried, ground to pass a 0.5mm
sieve, and stored in tight plastic jars for the analysis of nutrient
concentration and radioactivity. Soil samples collected from
each layer were placed in separate plastic bags, cleared of
visible roots and partially undecomposed residues, mixed
thoroughly, subsampled, air-dried, ground to ≤ 0.5mm, and
stored in plastic jars at room temperature prior to analysis.
chromium trioxide-acid mixture as described by Amato (1983).
Radioactivity (14C) of the soil sample was also measured
following acid digestion.
Extractable soil sulfate was measured based on the method
developed by Blair et al (1991). A 3-mL aliquot of extract was
taken for measurement of 35S activity.
Total carbon, 13C, total N and 15N concentrations were
analyzed using a Carlo-Erba NA 1500 elemental analyzer
coupled with a Europa Tracermass Isotope Ratio Mass
Spectrophotometer.
Chemical analyses
14
C and 35S were measured by digesting the plant materials
with Soluene-350 (Packard 1985) and counted in a Packard
TRI-CARB Liquid Scintillation Analyzer, Model 2000CA after
addition of 17 mL of liquid scintillation consisting of pterphenyl, POPOP, teric and toluene. A quench curve, consisting
of a series of spiked plant materials with varying amounts (5 to
25 mg) solubilized with 2 mL Soluene-350, was prepared to
correct for counting interferences due to Soluene-350 and to the
color resulting from the digestion. Since the beta spectra of 14C
and 35S and the region setting for 14C (0-156.48 kV) and 35S (0167.47 kV) 14C overlapped, it was difficult to calculate the
activities of the individual radionuclides using regional selection
settings as described for dual-labelled 3H and 14C in Packard
(1985). However, these two radionuclides vary greatly in terms
Calculations and statistical analyses
The proportion (%Ndfr) of compartment (soil or plant) N
derived from added crop residues was calculated using the
equation:
% Ndfr = (APEC ÷ APEAR) x 100
where APEC is atom% excess of compartment and APEAR is
atom% excess of added residue.
And the total amount of residue N (RDN) in the
compartment was calculated as:
RDN = [(compartment total N)(% Ndfr)]/100
The proportion of residue 14C or 35S derived from added
crop residue was calculated using the formula:
Proportion of residue 14C = (radioactivity of
compartment)/(radioactivity of added residue).
Data were subjected to analysis of variance using NEVA,
Analytical methods
Dry matter yield, nutrient concentration, 15N abundance and specific activity of Flemingia leaves and medic hay
produced
Table 1.
Plant
Material
Dry matter
(grams)
C (%)
Sp Act (kBq
14
C/gram C)
N (%)
Flemingia
185
48.24
38.29
Medic
155
45.45
39.62
of their half-lives: 5730 years for 14C and 87.39 days for 35S.
The same samples were then counted at different times and the
decrease in total radioactivity was attributed to the decay of 35S.
The following equations were used to calculate for radioactivity
of 14C and 35S using the counts obtained from two separate
dates:
At T1:Total Bq = Bq 14C + Bq 35S (1)
At T2:
Total Bq = Bq 14C + λ Bq 35S (2)
Where λ the decay factor is equal to:
λ = e −0.693(t/t1/2 )
To compare the use of Soluene-350 with acid digestion,
C in the plant materials was measured by digestion with
14
24
15
N (atom %)
S (%)
Sp Act (MBq
35
S/grams S)
3.34
1.66
0.20
27.80
4.45
2.53
0.35
38.64
following Burr (1982). Separation of means was performed
using Duncan’s Multiple Range Test (DMRT) at P = 0.05.
RESULTS & DISCUSSION
Production of labelled plant materials
A total of 185 grams of Flemingia leaves and 155 grams
oven-dry 14C-, 15N- and 35S-labelled medic hay were produced
from the first labelling. Details of the isotopic enrichments are
presented in Table 1. Flemingia leaves assimilated 9.2% of the
total 14C administered throughout the labelling period while
medic tops assimilated 7.5%, resulting in a total 14C uptake
Isotope-Labelling Of Plant Materials
efficiency of 16.7%. The overall labelling efficiency was not
estimated because the other plant parts of Flemingia (stems,
young leaves, roots) were not harvested since the same plants
were to be used in the succeeding labelling activities.
In the case of medic, only the aboveground biomass was
used. Voroney et al (1991) estimated their 14C labelling
efficiency at 50-80% and losses of incorporated label through
subsequent plant respiration at 30-60%. The amounts of 14C
Table 2.
Fate of residue C, N and S
The percentages of residue 14C recovered in the different
soil layers at various stages of decomposition are shown in
Table 3. For both residues, 14C recovery was highest in the top
soil layer and decreased at varying rates with time as
decomposition progressed. After 41 days of residue application,
almost half of the medic 14C had been released while 76% of
Flemingia 14C was still recovered in the top soil layer. At the
Dry matter, nutrient concentration and isotopic enrichment of Flemingia leaves, medic tops and rice straw
produced
Plant Material Dry matter
(grams)
C (%)
δ13C (‰)
Flemingia
230
44.14
Medic
150
Rice
500
13
Sp. Act
(kBq C/grams C)
N (%)
107.38
1.2291
13.09
3.45
1.69
43.65
117.01
1.2396
8.73
3.28
3.64
40.79
86.44
1.2061
7.33
1.62
2.10
incorporated into the plant tissue were similar for both
Flemingia (18.47 kBq 14C/gram Flemingia) and medic (18.01
kBq 14C/gram medic). indicating uniformity of labelling
between plants.
Medic tops contained higher 15N abundance and 35S
enrichments (Table 1) than Flemingia. Higher dry matter
production by Flemingia increased isotopic dilution, hence
lower 15N and 35S enrichments were obtained.
The second set of plant materials were grown for a longer
period and in summer, hence greater amounts of plant materials
were produced (Table 2). About 2.8% of the total 14C
administered was incorporated into the Flemingia leaves, 1.8%
into the medic tops and 2.6% into the rice straw. The 14C
labelling efficiency was lower than in the first set of plant
materials , which were less enriched with 14C but highly
enriched with 13C.
Feeding the plants with 14CO2 and 13CO2 at the same time
reduced the specific activity of 14C inside the chamber. This was
expected, as plants discriminate against the heavier isotope
(14CO2) during photosynthesis when they are supplied with both
13
14
CO2 and CO2 (Van Norman & Brown 1952). Rice straw
contained the lowest 13C and 14C enrichment due to
translocation to the grains, as below-ground translocation of
assimilated carbon is reduced (less than 5%) during the grainfilling stage (Keith et al 1986). Since the same Flemingia plants
that were used in the first labelling were re-exposed to 14CO2, the
Flemingia leaves contained more 14C compared to medic tops
and rice straw.
Pearl Basilio-Sanchez et al
15
C (atom
%)
14
N (atom %)
end of the experiment (108 days), 41.4% of medic 14C and
61.8% of Flemingia 14C were recovered. These values support
Jenkinson’s (1981) findings that about one-third of plant C
generally remains as organic residues after 1 year of
decomposition in the field.
Based on the amount of residue 14C recovered in the soil at
various stages of decomposition, the decomposition of medic
was significantly faster than Flemingia. The rapid decline in
medic 14C at early stages of decomposition suggests that medic
contains greater amounts of readily decomposable fractions of
plant C (Knapp et al 1983, Reinertsen et al 1984, Christensen
1985, Amato et al 1987, Ajwa & Tabatabai 1994).
A rapid decomposing residue like medic loses large
amounts of C during early decomposition stages and thus can
contribute only a small proportion to the soil organic pool. In
contrast, a slow and gradual release of C from Flemingia can
have a long-term effect in managing organic C level in the soil.
Higher 15N recovery in the soil was associated with the
application of Flemingia. About 50% of the 15N from the medic
residue application remained in the top soil layer while 77%
was recovered from the application of Flemingia (Table 3). The
amount recovered in the top layer declined with time in both
treatments as N uptake increased. The timely release of residue
15
N led to a 45% recovery of medic 15N in the millet, much
higher than the 13% recovery of Flemingia 15N after 41 days.
Medic contributed a higher amount of KCl-40 extractable
S in all soil layers (Table 3). At 41 days, 10% medic 35S was
present as extractable S in the top soil layer while only about 8%
25
of the Flemingia 35S was recovered in this layer. The ready
availability of medic 35S for plant uptake led to the recovery of
31% medic 35S at 41 days, which was much higher than the
13% recovery of Flemingia 35S. A higher supply of Flemingia
35S was, however, available for the second crop of millet.
CONCLUSIONS
The labelling chamber that was constructed was simple,
economical and effective in producing large quantities of multilabelled plant materials. Since it was constructed inside a
glasshouse, labelling could be done anytime of the year. The 14C
pulses used were minimal for safety precautions. The isotopic
enrichments attained were sufficient and enabled the
Table 3.
Recovery
14
C
15
N
35
S
decomposition process to be followed and the dynamics of the
residue-derived C, N and S to be studied. The use of the multilabelled plant residues provided a means of investigating
decomposition rate and C transformation, as well as quantifying
residues N and S taken up by the millet crop at various growth
stages.
Major differences were shown between the two types of
residues, medic hay and Fleminga, which have major
implications with respect to their usefulness as nutrient sources
and to the sequestration of carbon in soil. A plant residue with a
rapid decomposition rate like medic can provide an immediate
source of nutrients particularly for short-duration crops; a residue
that decomposes slowly like Flemingia results in a gradual release
of nutrients and hence can have longer impact on crop growth.
Proportion of residue 14C, 15N and 35S recovered (% of added) in the different soil layers at
various sampling times
Days After Addition
Soil
Layer
41
71
108
Flemingia
Medic
Flemingia
Medic
Flemingia
Medic
Top
76.0 a
56.1 b
73.9 a
42.2 b
59.6 a
39.3 b
Middle
1.6 c
1.2 c
1.0 c
1.5 c
1.5 c
0.7 c
Bottom
0.4 c
2.2 c
0.8 c
1.1 c
0.7 c
1.4 c
Top
77.1 a
55.4 b
74.5 a
53.0 b
71.7 a
51.0 b
Middle
0.5 c
1.8 c
0.7 c
2.7 c
2.7 c
2.5 c
Bottom
0.2 c
1.1 c
0.9 c
1.1 c
1.3 c
1.5 c
Top
7.65 b
10.16 a
4.05 a
4.17 a
4.70 a
3.64 a
Middle
1.11 c
1.30 c
0.37 b
0.52 b
0.41 b
0.27 b
Bottom
0.16 c
0.45 c
0.25 b
0.12 b
0.57 b
0.24 b
Within sampling time, means followed by the same letter are not significantly different at P = 0.05 by DMRT
Acknowledgment
This work was funded by the Australian Centre for International Agricultural Research (ACIAR) and conducted while the senior author
enjoyed an AusAID Scholarship at the University of New England, Armidale NSW, Australia. We are also grateful for the assistance of Dr
Anthony Whitbread, Mrs Leanne Lisle and Mrs Judi Kenny.
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