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.So;f Biol. Bimhem. Vol. 23. No. I?. pp. 110~1110. 1991 0038-0717/91 f3.00 + 0.

00
mntcd in Great Britain. All rights reserved Copyright C 1991 Pergamon Rtnc plc

EFFECT OF N FERTILIZER RATE ON THE ESTIMATION


OF N, FIXATION BY ISOTOPE DILUTION
S. D. HAMILTON,’ P. M. CHALK,‘* C. J. SMITH? and P. HOPMANS’
‘School of Agriculture and Forestry, University of Melbourne. Pdrkville 3052, 21nstitute for Sustainable
Development, Department of Agriculture, Tatura 3616 and ‘Natural Resources Division, Department of
Conservation and Environment. P.O. Box 406. Kew 3101. Victoria. Australia

(Accrprrd 28 July 1991)

Summary-Lupins and wheat fertilized with diRerent rates of “NH&I and K,“SO, (N:S = IO: 1) were
grown in the field in laterally-confined microplots. and the proportion of biologically-fixed N, in legume
tops (P,,,) harvested I91 days after sowing was estimated by the “N isotope dilution technique and the
A-value modification of this technique.
Estimates of P,,, decreased markedly as the rate of N applied to lupins increased from 2.5 to 20 kg
N ha-‘. irrespective of the rate of N applied to the reference plant, illustrating that symbiotic N, fixation
was inhibited by rates of N fertilizer commonly used to label soil.
Both nitrogen and sulphur A-values for wheat and lupins decreased as rates of fertilizer application
increased, and were associated with increases in efficiencies of fertilizer N and S use by the crops. Estimates
of P,,, generally increased as the rate of fertilizer N applied to the reference plant increased from 20 to
100 kg N ha-‘. but the extent of the increase was governed by the rate of N applied to the legume. Thus
the fundamental requirement of the A-value modification (i.e. the independence of the reference plant
A-value and rate of N application) was not fulfilled. and estimates of P,,, using A-values were higher than
cstimatcs using the classical methodology. It was concluded that the A-value modification of the “N
isotope dilution technique has only limited potential application in estimating the contribution of Nr
fixation to plant nutrition.

INTRODL’CTION The derivation of this equation was given by Fried


and Middclboc (1977).
The “N isotope dilution technique introduced by It is gcncrally believed that symbiotic Nr fixation
McAuliffe er ul. (1958) has been widely used to will be inhibited in soils of moderate to high N
cstimatc the proportion of N in plant material
fertility or by addition of fertilizer N. However,
derived from symbiotic or associative N: fixation interactions between rate of N addition, legume
(Chalk, 1985. 1991). The method depends upon cultivar and strain of Rhizobium have been demon-
differences in isotopic composition bctwccn the strated for soybean, and both positive and ncgativc
sources of N available to plants, i.e. soil N, fertilizer etfects of N on P,,, have been measured (Hardarson
N and atmospheric N,, and gcncrally involves er ul., 1984; Danso ef al., 1987; Senaratnc r~ al.. 1987).
addition of an “N-enriched fertilizer to the soil. Thus, The amount of legume dry matter production can
soil sources of N include the labelled (fertilizer) N moderate the effect of soil or fertilizer N on P,,,
and the indigenous soil N which is usually slightly (Evans er al., 1989). Thus addition of the isotope as
enriched in lJN compared to atmospheric N:. Un- inorganic N can have a direct effect on P&. but rates
labelled atmospheric NZ which is biologically fixed as high as 20 kg N ha-’ have frequently been used to
dilutes the labelled N taken up by the plant, and the label soil (Chalk, 1985).
measured isotopic dilution is used to estimate the A modification of the classical “N isotope dilution
proportion of legume N derived from the atmosphere method involving the addition of a higher rate of
(P,,,). The isotopic composition of the legume N N to the reference plant compared to the legume
derived from soil sources is usually estimated by was introduced by Fried and Broeshart (1975)-the
measuring the “N enrichment of a non-fixing rcfer- so-called “A-value” modification. The A-value is a
ence plant grown in soil labclled with N of the same measure of available N from unlabelled sources,
form and at the same rate and lJN enrichment as used which in the case of a non-fixing reference plant (A,,,)
with the legume. The principal assumption is that the is the soil and in the case of a legume (ALI) is the
isotopic composition of the N assimilated by fixing soil + atmosphere. The A-value (Fried and Dean,
and non-fixing plants from soil sources is the same 1952) is expressed as [BN (I - P&/P,], where
when intcgratcd over time, although the different B, = the amount of fertilizer N applied, P, = the
plants may assimilate ditTcrcnt amounts of soil and proportion of plant N derived from the fertilizer and
fertilizer N. P,,, is calculated as [I - (atom % “N (I - Pm) is the proportion of plant N derived from
excess of the legume material/atom % “N excess of unlabelled sources. The value for (I - Pe,) can be
the reference plant material)] (McAuliffe ef al., 19%). predicted for any rate of N fertilizer addition if the
measured A-value is independent of B,. In the appli-
*Author for correspondence. cation of this concept to the estimation of P,,,

SBB 21 I?--*
II05
1106 S. D. HAMILTON et of

the proportion of legume N derived from the labelled consisted of 2.5, 5, 10, 15 and 20 kg N ha-’ (lupins)
fertilizer source (P1,3 is measured and the proportion at 89.8, 44.9, 22.44, 14.96 and il.22 atom % “N,
of legume N derived from the unlabelled soil source respectively, and 2.5, 20, 50, 75 and 100 kg N ha-’
(P,,,,‘) is estimated as [(A,,) P,,/B,,J. The value of (wheat) at 89.8. 11.22,4.444,2.963 and 2.222 atom %
P,, is then calculated as [I - P,, - P,,,oll].Thus the “N, respectively. Sulphur was applied at 0.25, 0.5,
A-value modification of the “N isotope dilution 1.O, I .5 and 2.0 kg S ha- ’ (lupins), at specific activities
method is based on the independence of the reference of 740. 370, 185, 123.3 and 92.5 Bq pg-’ S, respect-
plant A-value (A,,,) from the rate of N application ively, and at 0.25, 2.0, 5.0, 7.5 and IO kg S ha-’
(B,,). (wheat) at specific activities of 740, 92.5, 37, 24.7
The A-value modification has often been used to and 18.5 Bq fig-’ S. respectively, The ten treatments
estimate P,,,. with the rate of N application to were completely randomized within each of four
reference plants being commonly 100 kg N ha-’ blocks.
(Chalk, 1985). The use of this modification has been The microplots were harvested at I91 days after
promoted by the International Atomic Energy sowing by removing all above-ground material. Plant
Agency (IAEA) in research involving legumes material was dried at 60‘C. weighed, milled to a fine
(Atkins, 1982). including co-ordinated research pro- powder and stored in sealed containers.
grammes of the Joint Division of FAO and IAEA
(e.g. Douka and Xenoulis. 1987; Ssali and Keya, Analytical
1983). However. few comparisons of estimates of P,,, Total N in plant material was determined by the
obtained by the classical technique and the A-value Kjeldahl digestion and steam distillation method
modification have been reported, and there are even of Nelson and Sommers (1973). Cross-contamination
fewer reports where the effect of Bcelon Amf was tested between ‘SN-labelled distillates was minimised by
experimentally. The objectives of the present study double distillation (Pruden et nl.. 1985). Nitrogen
were to determine the cffccts of increasing B,,, up to isotope ratios were measured with a dual-inlet, triple
20 kg N ha-’ on estimates of P,,, and of increasing collector mass spectrometer (VG lsogas Sira IO) on
Brcl up to IO0 kg N ha-’ on Arcl and estimates of P,,, NZ prepared from the oxidation of dried ammonium
using the classical and the A-value modification of distillates with alkaline hypobromite using the appar-
the isotope dilution tcchniquc when applied in the atus and procedures described by Chcn et al. (1990).
field to a grain Icgumc (lupins) and a cereal (wheat) Total S in plant material (80.-1OOmg) was dctcr-
as the rcfcrcncc plant. mined using the combustion and distillation pro-
ccdurcs dcscribcd by Bird and Fountain (1970).
>l,\TERIAI.S AND ~lKrlIoIEi Hydrogen sulphidc was trapped in IO ml of I M
NaOH and a 9 ml aliquot was titrated with 31.4 mM
mercuric acctatc using dithizonc-acctonc indicator. A
The field cxpcrimcnl was located at the lnstitutc I ml aliquot of the distillate was added to 9 ml of a
for Suslainablc Dcvclopmcnt, Taturn, Australia (lat. scintillation cocktail (Beckman Ready-GclT”) and
36’26’ S, long. I45 ‘15’ E, alt. II4 m). The soil at the counted on a Beckman LS 3801 liquid scintillation
silt is described as a red-brown earth (State CI ui., counter, following calibration of the instrument
1968) or a Typic Haplustalf (SMSS. 1983). The using carbon tctrachloridc quenching (Long, 1976).
profile is charactcriscd by a loam A horizon to I5 cm Mcasurcd spccitic activities were standardized to zero
depth (pH 5.4, I: 5 soil: 0.1 M CaCI,!. with an abrupt time on the basis that the half-life of “S = 88 days.
boundary to a clay B horizon cxtcnd’ng to 60 cm. The Proportions of plant N derived from the fcrtilizcr
A horizon soil had a cation exchange capacity of (Pm) were dctermincd as (atom % “N excess of the
12.4cmol (+) kg-’ and total N and organic C plant material/atom % “N excess of the fertilizer)
contents of 1.60 and 17.8 g kg-‘, respectively. using 0.366 atom % “N as natural abundance.
The site was cultivated to a depth of IOcm prior Proportions of plant S derived from the fertilizer (P,*)
to sowing on 25 April 1988. Treatments consisted of were determined as (specific activity of the plant
two crop species, lupins (Lupinus angusfi/blius L. cv. material/specific activity of the fertilizer). Procedures
Yandce) and wheat (Triricum aesfium L. cv. Ma- used for calculating nitrogen A-values and pro-
tong), fertilized with different rates of nitro- portions of plant N derived from the atmosphere
gcn + sulphur at a constant ratio of N: S of IO: 1. Plot (P,,,) were described in the Introduction to this
size was 2 x 7 m. Wheat and lupins were sown at article. VillUeS of P,,, were calculated only for
rates of 120 and 150 kg seed ha-‘, respectively, in treatment combinations where Brd > B,,,. Sulphur
drill rows spaced at 15cm. Lupin seed was slurry- A-values = [B, (I - P&/P,], where B, = the amount
inoculated with a commercial peat-based inoculant of fertilizer S applied. The proportions of applied
(Rhixthiutn hrpini WU425) at 2.5 g kg-’ seed immedi- fertilizer N or S in harvested plant material, i.e.
atcly prior to sowing. the cfficicncics of fcrtilizcr USC (E) were calculated
A laterally-confined microplot was established as E, = (N,,,,,) Pm/B, or Es = (Splm,) P,/B,.
within each plot following sowing by driving an respectively. Data were analysed using the analysis of
open-ended steel chamber (30 x 30 x 30 cm) centered variance procedures of Gcnstat 5 (Payne, 1987).
on the row to a depth of 25 cm. ‘5N-labelled NH,CI
and “S-labclled K$O, were uniformly applied to RESULTS AND DISCUSSION
each microplot by spraying individual solutions onto
the soil surface. Five rates of N + S were applied Effect of rate of fertiker addirion on A-values
to each species but only two rates were common to Nitrogen A-values for both wheat and lupins de-
both lupins and wheat. Nitrogen fertilizer treatments creased as the rate of N fertilizer addition increased,
Estimation of Nr fixation by isotope dilution 1107

with the decrease being most pronounced between the (a)


two lowest rates of N application (Fig. I). Smith et al.
(1989) also measured decreasing nitrogen A-values at
this site with increasing amounts of N applied to
barley, and showed that the decrease in nitrogen 0.6’
A-values was associated with an increase in the EN 0.5.
efficiency of N fertilizer use (E,) as N rate increased.
0*-
Similarly, decreases in nitrogen A-values in the pre-
sent experiment were related to increasing pro- 03’
portions of fertilizer N uptake by both wheat and 0.2-
lupins as N rate increased (Fig. 2). Sulphur A-values 0.1-
for both wheat and lupins also decreased as S fertil- co-!, U, I , I
izer rate increased (Fig. I). and this too was associ- 0 IO 20 50 75 loo
ated with increasing efficiencies of fertilizer S use as N fertilizer (kg N ho-‘)
S rate increased (Fig. 2). Thus the trends in A-values 1.0,
for sulphur were similar to those obtained for nitro- W (b)
09’ L
gen for increasing rates of fertilizer additions. I t
0.8-
In other field studies with grain legumes in which
0.7-
the A-value modification was used to estimate P,,, it
was also found that the A-value of the reference plant 06-
decreased with increasing rate of N applied (V&as Es 0.5-
and Ham. 1984; Smith rt al., 1987). The decrease 0.J-
in A-value for non-nodulating soybean occurred 0.3-
between 5 and 100 kg N ha-’ and was consistent over 0.2-
seasons, sites and stage of plant growth (Vasilas and 0.1-
Ham, 1984). Similarly, Smith fr ul. (1987) found that I I I I ,
the A-values for two rcfcrcncc plants (wheat and 0 I 2 5 75 IO
non-nodulating chickpea) consistently dccrcascd over
S fertilizer (kg S ho-‘)
stxson and site when the N rate was incrcascd from
I8 to 72 or from IO to 80 kg N ha ‘. The dcpcndcncc Fig. 2. Effect of rate of addition of (a) “NH,CI and (b)
K,“SO, on the proportions of fertilizer N (E,) and S (E,)
rccovercd in the tops of wheat (open symbols) and lupins
(solid symbols) harvested 191 days after sowing. Vertical
(a) bars represent lcast significant diffcrcnccs (P = 0.05) for
wheat (W) and lupins (L) for comparisons between fertilizer
treatments.

of A,,,. on BCclcauses a dilemma in the estimation of


P ,,,,,, i.e. whrch value of Arer should be used to
calculate P,,,? Smith et al. (1987) used the higher N
rate when the reference plants responded to N and
the lower N rate when there was no response, but
recognised that a sound basis for deciding which
z ot,,,,, I I estimate was more accurate was lacking.
0 IO 20 SO 75 1(X1 Thus the results of the present investigation and
N fertilizer (kg N ha“) other field studies with legumes have shown that
A-values of reference plants are not independent of
(h)
the amount of fertilizer N added as was suggested on
several occasions by Fried and Broeshart (1975).
Both theoretical evidence (Jenkinson ef al., 1985) and
experimental results (Hart et al., 1986) have been
presented to show that A-values are dependent on N
fertilizer rate due pool substitution brought about by
immobilization of the lJN-labelled fertilizer. Thus,
A-values are most likely to be independent of N
fcrtilizcr rate in coarse-textured soils low in organic
matter and biological activity, or when biological
activity is suppressed due to soil fumigation (e.g.
0 I 2 5 7.5 IO Gauthicr et al.. 1985; Sougoufara ef al.. 1990).
S fertilizer (kg S ho-‘)
Eflecr of B,, on esrimates of P,,_
Fig I. EtTcct of rate of fertilizer addition as (a) “NH,CI and
(b) K,%O, on nitrogen and sulphur A-values for wheat
Estimates of P,,, were independent of the rate of
(open symbols) and lupins (solid symbols) harvested I91
N applied to wheat between 20 and 50 kg N ha-’
days after sowing. Vertical bars represent least significant irrespective of the amount of N applied to lupins, and
differences (P =0.05) for wheat (W) and lupins (L) for were independent of the amount of N applied to
comparisons between fertilizer treatments. wheat between 20 and 100 kg N ha-’ when the lowest
1108 S. D. HAMILTON CI al.

N applied to lupins (kg N ha-‘) Exation was inhibited by the labelled N fertilizer.
Previous work (Evans et ul., 1987) indicated that
the lupin-Rhizobium symbiosis was insensitive to N
fertilizer addition, but subsequent experiments
(Evans et 01.. 1989) demonstrated an inverse relation-
ship between P,, and available soil N which was
attenuated by increasing lupin dry matter production.
0.6
Thus, it appears that the amount of fertilizer N used
P (Itm to label legume plots should be considerably less than
OJ 20 kg N ha-’ which has frequently been applied in
past experiments with grain legumes (Chalk, 1985).
0.2 However, results obtained in the present investigation
and model predictions of Jenkinson et al. (1985) show
001 25 20 so
_.
75 loo
that at low rates of fertilizer addition A-values are
sensitive to small changes in B,. Therefore, the
N applied to wheat (kg N ha“)
A-value modification seems to be incompatible with
the need to keep B,,, low yet at the same time fulfill
Fig. 3. Relationships between estimates of the proportion of
the requirement for A,r to be independent of B,,.
legume N (P,,,) derived from the atmosphere at 191 days
after sowing and the rate of “NH&I applied to wheat at
Calculation of P,,,
different rates of N applied to lupins. Open and solid
symbols represent values calculated by the classical and The proportion of biologically fixed N in harvested
A-value modification of the “N isotope dilution technique, legume material (P.,,) was calculated as [I - P’, -
respectively. Vertical bars represent least significant differ- (A,,,P,,,/B,,,)] when the rate of N added to the refer-
ences (P = 0.05) for comparisons between N treatments ence plant (B,,) was higher than that added to
applied to wheat. the legume (B,,,). Several mathematically-equivalent
expressions have also been used to calculate P,,,, e.g.

rate of N (2.5 kg N ha-‘) was applied to lupins P.,, = [(A’,, - A,,r)Pl,,/B’,J (Danso, 1988)
(Fig. 3). Estimates of P,,, increased markedly be-
or
tween 50 and 100 kg N ha-’ applied to wheat when
the amount of N applied to lupin was bctwecn 5 and P,,, = { 1 - PI<, - h PI,, (1 - P,,,YP,,~I~Frid 1985)
20 kg N ha-‘, and when 2.5 kg N ha-’ was applied to
lupin P,,, increased markedly bctwcen 2.5 and 20 kg where
N ha-’ applied to wheat (Fig. 3). Thus cstimatcs of
n = BrcflBlep.and P,, = the proportion of reference
P,,, gcncrally increased as the rate of fcrtilizcr N plant N dcrivcd from the fertilizer.
applied to the rcfcrcnce plant increased. but the
extent of the increase was governed by the rate of N Prior to the publication of the expression by Fried
applied to the legume. Therefore estimates of P,,, by (1985) there was controversy in the literature regard-
the A-value modification were higher than estimates ing the yield-dependence or yield-independence of
by the classical lJN isotope dilution method (Fig. 3) estimates of NZ fixation using the A-value modifi-
with P,,, increasing from 0.56 to 0.96 (B’,, = 2.5 kg N cation of the lJN isotope dilution method. This arose
ha-‘) and from 0.22 to 0.52 (B,,, = 20 kg N ha-‘). because the original A-value expression was for the
Estimates of P,,, obtained by the classical and the amount of biologically-fixed N in harvested material,
A-value modification of the isotope dilution tech- which is yield-dependent, i.e.
nique have been compared in several studies, but
comparison of data obtained in pot studies (Ma et al., N, fixed = [(A’,, - A,,,) J&l
1984, 1987; Sanginga ef al.. 1990) where roots are (Fried and Broeshart, 1975)
totally confined, or comparisons using fumigated soil
(Gauthier er al., 1985; Sougoufara ef al., 1990) where Had these authors simultaneously provided an ex-
biological processes are inhibited are unlikely to pression for Pa,,, which is independent of yield, it is
provide results which are characteristic of A-values unlikely that the controversy would ever have arisen.
determined in the field under natural conditions. In We conclude that the use of the A-value modifi-
all but one of the comparisons made with field data cation as a strategy for improving the accuracy of
it was shown that estimates of P,,, determined using field estimates of P,,, for legumes cannot be rec-
A-values were higher than values determined by the ommended. It is not only subject to the same errors
classical procedure (Vasilas and Ham, 1984; Smith et encountered with the classical procedure due to the
al.. 1987; Papastylianou and Danso, 1989) which is non-uniform distribution of the isotope, but it has the
consistent with results obtained in the present study. additional requirement of the constancy of A,, when
Smith er al. (1987) suggested that P,,, may be over- higher rates of N are applied to the non-fixing plant.
estimated using the A-value modification. The results of the present investigation and other field
studies with grain legumes do not generally support
Eflecf o$ B,, on estimates of P,,‘_, this latter requirement. The use of labelling treat-
Estimates of P,,, decreased markedly as the rate of ments which lead to a more stable lJN enrichment in
N applied to lupins increased from 2.5 to 20 kg N the available soil N pool is a strategy which has been
ha-‘, irrespective of the rate of N applied to the recommended for improving the accuracy of field
reference plant (Fig. 3), illustrating that symbiotic N, estimates of P,,, (Witty, 1983).
Estimation of N, lixa ition by isotope dilution 1109

The A-value modification also lacks application in Fried M. and Dean L. A. (1952) A concept concerning the
mixed stands of legumes and reference plants. How- measurement of available soil nutrients. Soil Science 73,
ever, it appears that the modification may have a 263-27 !
potentially useful application in the estimation of N, Fried M. and Middelboe V. (1977) Measurement of amount
of nitrogen fixed by a legume crop. Pfunr und Soil 47,
fixation associated with cereals and C4 grasses
713-715.
(Chalk, 1991). where low rates of N, hation could be Gauthier D., Diem H. G., Dommergues Y. R. and Gamy
suppressed by addition of N fertilizer. By applying F. (1985) Assessment of N, fixation by Caruorina equiwti-
fertilizer N at a very low rate and at a rate high folio inoculated with Frunkiu ORSOZIOOI using 15N
enough to effectively inhibit associative N, fixation, methods. Soil Biology & Biochemisrry 17, 375-379.
fixing and non-fixing treatments with the same plant Hardarson G., Zapata F. and Danso S. K. A. (1984) Effect
species are obtained. The technique has been used to of plant genotype and nitrogen fertilizer on symbiotic
estimate N2 fixation associated with field-grown nitrogen fixation by soybean cullivars. Plant and Soil 82.
kallar grass (Malik et al., 1988). and the use of 397-405.
Hart P. B. S., Rayner J. H. and Jenkinson D. S. (1986)
uninoculated (-N), uninoculated (+ N) and inocu-
Influence of pool substitution on the interpretation of
lated (-N) treatments permitted estimation of puta-
fertilizer experiments with “N. Journal of Soil Science 37.
tive N, fixation due to natural and inoculated 389403.
sources. Jenkinson D. S., Fox R. H. and Rayner J. H. (1985)
Interactions between fertilizer nitrogen and soil nitro-
Acknowlcdgemenfs-We thank the Reserve Bank of Aus- gen-the so-called ‘priming’ effect. Journal ofSoil Science
tralia for financial support from the Rural Credits Develop- 36, 425444.
ment Fund. and Mr D. Chen for translation of the articles Long E. C. (1976) Liquid scintillation counting theory
by Ma ef al. (1984, 1987). and techniques. Technical Report No. 915-NUC-76-7T,
Beckman Instruments. Irvine.
Ma C. L.. Wen X. F., Yao Y. Y., Zhou C. X. and Liu X. L.
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