Chapter 13

Biological Nitrogen Fixation

K.E. GILLER

Plant Production Systems, Department of Plant Sciences,

Wageningen University, PO Box 430, 6700 AK Wageningen, The
Netherlands

13.1 Synopsis

Trees can increase nutrient inputs and reduce nutrient losses through a
variety of processes. Biological nitrogen fixation is prominent among these
as it is the only truly renewable source of nutrients in agroforestry or
agriculture as a whole. Many of the trees used in agroforestry are legumes
as the ability to fix nitrogen allows them to grow rapidly in nitrogen-
depleted soils. Thus one of the principal roles of nitrogen-fixing trees in
agroforestry is to improve soil fertility, athough many legume trees have
multiple uses for fodder, fuelwood, fruits and timber. Legume trees are
thus found in almost all types of agroforestry systems: as shade trees in
perennial crops, in improved fallows, in hedgerow intercropping systems,
as erosion barriers, live fences, isolated trees in parklands and fodder trees.
Not all legumes can nodulate and fix nitrogen, and the taxonomy of
the Leguminosae is a good guide to which legume trees are nitrogen fixers.
Nitrogen-fixing symbioses are also formed between non-legume trees and
actinomycetes (Frankia spp.), which are termed actinorhizal symbioses
(Giller, 2001). The most important of these are the Casuarina spp. from
Australasia, which are used for soil stabilization, as windbreaks and for
poles and fuelwood throughout the tropics. The actinorhizal symbioses
have been less studied in agroforestry than leguminous trees, but the
methods outlined here can equally well be applied to the study of either
type of symbiosis. A full list of nitrogen-fixing trees also includes larger
species of cycads, which form symbioses in their leaf axils with blue-green
algae (Cyanobacteria), but these are of no importance in agroforestry.

© CAB International 2003. Trees, Crops and Soil Fertility (eds G. Schroth and 259
F.L. Sinclair)
260 K.E. Giller

Nodulation and nitrogen fixation in the Leguminosae

The Leguminosae are a large and diverse family of flowering plants

composed of some 18,000 species, ranging from huge, long-lived forest
trees to tiny, annual, herbaceous forbs. All legumes have nitrogen-rich
tissues, irrespective of whether they can fix nitrogen, and it is now thought
that the nitrogen fixation ability evolved to satisfy the demand for nitrogen,
rather than nitrogen fixation resulting in the high tissue concentrations
of nitrogen, which had often been assumed earlier (see McKey, 1994;
Giller, 1997). Those legumes, which are not able to fix nitrogen, also have
a large tissue nitrogen demand, which they satisfy either by competing
actively for soil nitrogen or by growing more slowly and conserving their
nitrogen within the plant. A further major consequence of the large tissue
concentrations of nitrogen in the Leguminosae is the wide variety of
secondary metabolites which are produced to protect legume tissues from
predation (Waterman, 1994). Among these secondary metabolites the
polyphenols, and particularly the condensed tannins, play a major role in
regulating nitrogen release from legume tissues in decomposition (see
Sections 6.1 and 6.4) and during ruminant digestion (Giller, 1997).
The Leguminosae are divided into three subfamilies, the Caesalpinioideae,
the Mimosoideae and the Papilionoideae, and these subfamilies provide a
useful guide as to whether the legumes are able to nodulate and fix
nitrogen. The Caesalpinioideae, of which only 23% form nodules (de Faria
et al., 1989; Sprent, 2001), are considered to be the most primitive group.
Within the Caesalpinioideae, nodulation is restricted to a few tribes, the most
notable of which is Cassieae, which appear to form a bridging group
between the non-nodulating and nodulating legumes. Within this tribe all
members of genera Cassia (sensu stricto) and Senna are unable to nodulate.
The genus Senna includes several important agroforestry species, such as
S. siamea and S. spectabilis, which have been widely tested in hedgerow
intercropping and were earlier assigned to the genus Cassia (Irwin and
Barneby, 1981). Closely related to the genera Cassia and Senna is the genus
Chamaecrista, in which all the species examined nodulate and fix nitrogen.
The range of nodule morphology within the genus Chamaecrista is striking,
with some species having rather primitive nodule structures, in which the
bacteria are maintained in persistent infection threads within the nodule
cells, whereas in other species fully differentiated bacteroids are released
within peribacteroid membranes in the host cell cytoplasm (Naisbitt et al.,
1992). Persistent infection threads are also found in the only non-legume
tree genus, Parasponia (Ulmaceae), which can form nodules and fix nitrogen
with rhizobia (Trinick and Hadobas, 1988; Becking, 1992).
Almost all legumes in the other two subfamilies, the Mimosoideae (90%)
and the Papilionoideae (97%), can form nodules with rhizobia and fix
nitrogen (de Faria et al., 1989). The exceptions in the Mimosoideae appear
Biological Nitrogen Fixation 261

to be cases where loss of nodulation and nitrogen-fixation ability has

occurred in species which are found in very arid environments (e.g. some
Acacia spp.), presumably due to the sensitivity of nitrogen fixation to
drought (Odee and Sprent, 1992; Sprent, 1994). Members of the
Papilionoideae which do not nodulate are largely those found in primitive
tribes.
Useful reference texts with information on nodulation and nitrogen
fixation in legumes are Allen and Allen (1981) and Sprent (2001).

Other sources of nitrogen fixation in agroforestry systems

Apart from inputs from nitrogen-fixing trees, herbaceous cover crops/

green manure legumes and grain legumes are often important com-
ponents of agroforestry systems and further information can be found in
Giller (2001). The discovery of endophytic nitrogen-fixing bacteria in the
tissues (particularly the vascular tissues) of a range of non-legume species,
including oil palm (Elaeis guineensis), has led to claims that such bacteria
may give substantial inputs of nitrogen. Direct evidence for substantial
nitrogen inputs (~20 kg N ha–1 year–1) is strongest in sugar cane (Saccharum
officinarum; e.g. Boddey, 1995), though the evidence is still controversial.
Nitrogen inputs from heterotrophic, free-living nitrogen-fixing bacteria
in soil are generally considered to be very limited and not of significance
except over long time scales (Giller and Day, 1985; Vanderleyden, 1997;
Giller, 2001).

Environmental limitations to nitrogen fixation

All environmental limitations that adversely affect plant growth and vigour
also decrease amounts of nitrogen fixation in legumes, although the
symbiosis is sometimes more sensitive to such constraints than other aspects
of plant growth (Giller, 2001). Nitrogen fixation is sensitive to nutrient
deficiencies, in particular phosphorus deficiency, which may restrict
nodule formation if acute. Molybdenum deficiency influences nitrogen
fixation directly as molybdenum is a component of the nitrogenase
enzyme. Nitrogen fixation is thought to be more sensitive to drought stress
than other processes, such as photosynthesis, although the evidence is
somewhat equivocal (Sprent, 1984). A further interesting feature of the
legume–rhizobium symbiosis is that the sensitivity to stress may be
expressed through the bacteria, the legume host or the formation of the
symbiosis itself. Legumes are invariably more sensitive to salinity than are
rhizobia (Sprent, 1984), but rhizobia are more sensitive than their hosts to
heavy metal pollution (Giller et al., 1998). The infection process itself
262 K.E. Giller

appears to be particularly sensitive to calcium deficiency (Giller, 2001).

Large differences in sensitivity to stresses such as aluminium toxicity in
soil are found among rhizobial strains and among legume hosts.

Effects of management on nodulation and nitrogen fixation

Legume trees are often pruned severely in agroforestry systems to provide

fodder or foliage for soil amendment. It is well established that defoliation
causes nodule senescence (Witty and Minchin, 1988), and pruning or
browsing of trees by animals causes temporary decreases in the rates of
nitrogen fixation. Reestablishment of nitrogen fixation will depend on
formation of new nodules, though this can sometimes be rapid as legumes
often harbour latent infections in young roots, which can develop when
nitrogen demand in the plant is large. However, in Erythrina poeppigiana
pruning resulted in complete mortality of nodules and there was a lag of
10 weeks before active nodules were re-formed (Nygren and Ramírez,
1995). Similarly, pruning of Leucaena diversifolia resulted in drastic
reduction of nodule activity (measured as acetylene reduction) for about
3 months (Snoeck, 1995). Nygren (1995) highlighted the danger that
nodulation might be completely suppressed if trees are pruned too
frequently.
Nodule senescence associated with defoliation of trees is a mechanism
by which nitrogen is made available to plants growing in close proximity,
though amounts of nitrogen made available in this way are likely to be
relatively small (Rao and Giller, 1993; Nygren and Ramírez, 1995). Even

Table 13.1. Mechanisms by which nitrogen from nitrogen-fixing trees can be

made available to other plants.a

Rate of Likely importance

Mechanism transfer as an N source
Below ground
Root and nodule senescence and mineralization Slow Major
Rhizodeposition Rapid Minimal
Transfer between roots by interconnected
mycorrhizal hyphae Rapid Minimal
Above ground
Mineralization of severed or senesced plant material Slow Major
Consumption by grazing animals or insects and
return in excreta or as carcasses Slow/rapid Major
Foliar leachates Rapid Moderate
Transfer of ammonia to associated plants Rapid Minimal
aModified from Ledgard and Giller (1995).
Biological Nitrogen Fixation 263

Box 13.1. Rhizobia that nodulate legume trees.

The rhizobia that nodulate legume trees are a sadly neglected area of study
compared with the amount of research effort which has been directed to grain
legumes. The taxonomy of rhizobia has developed rapidly since the advent of
phylogenetic methods for bacterial classification based on sequence analysis of
the 16S rRNA gene (Young and Haukka, 1996). The older classification methods
were largely based on the legume host range of the rhizobia. Although this
approach was recognized to be severely flawed early on (Wilson, 1944), the new
classification reveals the true extent of such problems in that nodulation ability is
closely related to the nodulation genes carried by the rhizobia, rather than their
evolutionary similarity (Young and Haukka, 1996). The nodulation genes are
generally carried on transmissible plasmids in fast-growing rhizobia, and in some
cases on transmissible ‘symbiotic islands’ of chromosomal DNA (Sullivan and
Ronson, 1998). Rhizobia far apart on the phylogenetic tree may thus carry the
same nodulation genes and have a very similar host range for nodulation and
nitrogen fixation.
Research in the last 10 years has revealed some surprising overlaps between
rhizobia which can nodulate legume trees, herbaceous and grain legumes. The
best documented case is that of Rhizobium sp. NGR234, which has been shown
to effectively nodulate legumes from 112 genera, including members of the three
different subfamilies of the Leguminosae (Pueppke and Broughton, 1999). Other
broad-host-range rhizobia that nodulate trees are Rhizobium tropici (Martínez-
Romero et al., 1991), which nodulates Leucaena spp. and Phaseolus vulgaris, and
indeed other rhizobia species such as R. etli, which were thought to be specific
in their host range, have since been shown to nodulate a wider range of species
including trees (Hernandez Lucas et al., 1995).
Slow-growing rhizobia are all members of a single genus, Bradyrhizobium,
whereas the fast-growing rhizobia have been split into several genera. Five genera
of fast-growing rhizobia are currently recognized: Rhizobium, Azorhizobium,
Sinorhizobium, Mesorhizobium and Allorhizobium, although this field is changing
rapidly. Some trees are reported to nodulate only with slow-growing rhizobia
(Dreyfus and Dommergues, 1981) but the vast majority examined nodulate with
fast-growing rhizobia. Of particular note are the rhizobia which form stem nodules;
shrubby legumes of the genus Aeschynomene form stem nodules with
photosynthetic slow-growing Bradyrhizobium strains, whereas Sesbania rostrata
forms nodules with rhizobia classified into a separate genus, Azorhizobium
caulinodans (Giller, 2001).
Recent studies of rhizobia that nodulate legume trees have resulted in the
description of the new species Sinorhizobium terangae and Sinorhizobium saheli
from nodules of Sesbania and Acacia spp. in Senegal (de Lajudie et al., 1994) and
Mesorhizobium plurifarium (de Lajudie et al., 1998), Sinorhizobium arboris and
Sinorhizobium kostiense (Nick et al., 1999) from nodules of Prosopis and Acacia
spp. in Sudan and Kenya. The classification has recently been substantially revised
(see Young et al., 2001)
264 K.E. Giller

with E. poeppigiana, which was profusely nodulated in the field, amounts

of nitrogen released from complete senescence of the nodules were much
less than 10 kg N ha–1 (Nygren and Ramírez, 1995). If the turnover of fine
roots is included, the amounts of N recycled below ground are likely to be
of considerable importance but accurate estimates are lacking. The major
pathway by which nitrogen is made available for other plants is through
recycling of above-ground litter either directly to the soil, or through
manure of animals grazing on the trees (Table 13.1).

Requirements for inoculation

The success of many of the fast-growing legume trees in agroforestry has

undoubtedly been due to their ability to nodulate and fix nitrogen in soils
across the tropics. Unfortunately, it is almost impossible to generalize as
to which species are likely to nodulate readily in soils where they have not
been grown, although there are gradations of the degree of promiscuity
or specificity among agroforestry trees (Bala and Giller, 2001). Bala (1999)
hypothesized that the success of species such as Leucaena leucocephala and
Gliricidia sepium could have been due to either: (i) their ability to nodulate
promiscuously with a wide range of rhizobial types, or (ii) the fact that,
although such trees may nodulate with a narrow range of rhizobial species,
these species are ubiquitous throughout the tropics. To test these
hypotheses he characterized rhizobial isolates from nodules of Leucaena
leucocephala, Calliandra calothyrsus, Gliricidia sepium and Sesbania sesban
grown in soils from Central and South America, West, East and southern
Africa and South-east Asia. Rhizobial isolates were characterized on the
basis of physiological tests, host range for nodulation and molecular typing
methods (including 16S rRNA partial gene sequences). Bala (1999) found
that rhizobial isolates nodulating Leucaena, Calliandra and Gliricidia were
diverse, belonging to a wide range of strains of the genera Rhizobium,
Mesorhizobium, Sinorhizobium and Allorhizobium/Agrobacterium. This supports
the first hypothesis that the tree legumes nodulated promiscuously with a
wide range of rhizobia. Further, these three tree legumes nodulated with
many of the same isolates although there were isolates which nodulated
with only one or two of the species. Sesbania sesban was an exception in that
it nodulated with a largely separate range of isolates, which fell into the
Rhizobium and Mesorhizobium genera. Even more surprising was the limited
range of soils from southern Africa in which S. sesban nodulated,
considering that it is naturally found in this region. This clearly indicated
that S. sesban requires inoculation when sown for the first time into upland
soils. Details of how rhizobial inoculants can be made and applied are given
in the methods manuals cited below.
Biological Nitrogen Fixation 265

13.2 Microbiological Methods for Studying Rhizobia

Methods for the isolation of rhizobia from nodules and authentication are
simple and can be conducted in any laboratory which has the capacity for
simple microbiology. There are a number of excellent methods manuals
including Vincent (1970), Somesagaran and Hoben (1985) and Sylvester-
Bradley and Kipe-Nolt (1988). Host-range studies provide information on
the ability of individual isolates to nodulate and fix nitrogen with different
legumes, and therefore can give some clues as to the phylogenetic
relatedness of strains, but detailed strain characterization now relies on
methods of molecular biology, which tends to restrict such activities.

13.3 Simple Methods for Determining Whether a Legume is

Fixing Nitrogen
In the absence of access to isotope-based methods (see below) there are
some simple approaches to assessing whether trees or crops are benefiting
from nitrogen fixation.
• The simplest and most obvious (though often overlooked) approach
to determine whether a tree is fixing nitrogen is to excavate the root
system and look for nodules. If nodules are found, then this is strong
evidence that the trees are or have recently been fixing nitrogen. If
no nodules are found this could be due to a number of reasons: that
the root excavations are not complete and the nodules are only found
in deeper horizons, or that the nodules readily become detached from
the roots and lost during sampling, which is particularly problematic
with some species (e.g. Cajanus cajan, Gliricidia sepium).
• The ability of a tree to grow and accumulate nitrogen in infertile or
skeletal soils may be evidence of nitrogen fixation. Sanginga et al.
(1986) compared nitrogen accumulation in prunings of inoculated and
uninoculated Leucaena leucocephala and estimated that 224–274 kg N
ha–1 came from nitrogen fixation. This approach to estimating
nitrogen fixation, which is a simple and useful approach to estimating
nitrogen benefits, is referred to as the nitrogen balance method.
However, in many situations it is dangerous to rely on such evidence
alone as tree roots might be accessing deep soil horizons richer in
nitrogen (see Box 8.1 on p. 171), or there may be subsurface lateral
flow of water from which the trees can scavenge nitrogen. A further
development of this approach is to develop a whole nitrogen balance
for the system, preferably comparing a system with nitrogen-fixing
trees with a system with non-nitrogen-fixing trees. Such studies suffer
from the large number of unknown processes such as gaseous losses,
266 K.E. Giller

which are as difficult to estimate as nitrogen fixation. A primary

advantage of agroforestry systems is that the trees are able to access
nutrient sources which are not available to other plants, which in itself
can cause problems in calculations of nitrogen balances if nitrogen is
taken up from deep water tables.

13.4 Isotope-based Methods for Measurement of Nitrogen

Fixation
15N-isotope-based methods are perhaps the only means available for
estimating amounts of nitrogen from nitrogen fixation in trees growing in
the field, though such methods may have significant problems in their
application. The two main variants of these methods are those involving
addition of 15N-labelled fertilizers (often called 15N-isotope dilution
methods) and those that rely on the natural 15N-enrichments of soil (known
as natural abundance methods). Most soils are naturally, slightly enriched
with 15N with δ15N values commonly of 2–16‰ (the difference between the
15N content of the material and air expressed in parts per 1000):

 Rsample – Rreference 
δ15N (in parts per 1000 or 0/00) =   × 1000 (13.1)
 Rreference 
where during chemical analysis R is the ratio of N2 molecules derived from
the plant material, which are composed of one 15N and one 14N atom to
those composed of two 14N atoms, i.e.
15
N+ 14 N
R= (13.2)
14
N+ 14 N
The basic principle of both methods is that nitrogen taken up from
the soil is enriched with the heavy isotope 15N compared with nitrogen
absorbed from the atmosphere. A non-nitrogen-fixing reference plant is
used to estimate the 15N-enrichment of nitrogen available from the soil in
both cases (Peoples et al., 1989). The major assumption (and problem) of
these methods is that the 15N-enrichment of the available soil nitrogen
taken up by the reference plant is the same as that taken up by the
nitrogen-fixing legume. This is in fact only true when there is a perfectly
uniform 15N-enrichment of plant available nitrogen in the soil with depth
and time, or, if this is not the case, when the rooting depths and activity
and the time course of nitrogen uptake of the reference and legume plants
are identical (Witty, 1983). This assumption is rarely satisfied and therefore
steps must be taken to limit the variability in 15N-enrichment of the
available soil nitrogen as far as possible (Witty and Giller, 1991). The use
of direct measurements of the 15N-enrichment of the available soil nitrogen
Biological Nitrogen Fixation 267

coupled with modelling have been recommended as an alternative to using

reference plants, but such approaches require extensive sampling and
analysis of extractable soil nitrogen (Chalk and Ladha, 1999) and are
unlikely to be widely used. Much has been written on the problems of
measuring nitrogen fixation using isotope approaches (e.g. Witty, 1983;
Witty and Giller, 1991; Chalk and Ladha, 1999). Problems encountered
with herbaceous legumes when isotope methods are applied are magni-
fied in studies with trees due to their much larger size and longevity
(Boddey et al., 2000). The size of trees and lignification of plant parts
presents additional problems of subsampling of plant tissues to give a
representative sample for measurement of tissue nitrogen concentrations
and 15N-abundance.

Box 13.2. Measuring nitrogen fixation under controlled

conditions.
There are remarkably few measurements of nitrogen fixation by nitrogen-fixing
trees growing in the field. This is in no small part due to the technical problems
that exist in the application of the available methods, and partly for this reason
many measurements have been confined to studies under more controlled
conditions with trees grown in pots. The major limitations of these pot studies is
that they do not often provide the information we need to assess the role of nitrogen
fixation in agroforestry systems.
So what do we want to measure? Do we want to know which of the different
provenances of Gliricidia sepium fix the most nitrogen after 6 weeks of growth in
a glasshouse? Or do we wish to pose the question: are all rhizobial strains that
belong to different species or genera equally efficient in nitrogen fixation with a
particular tree species? If these are the types of questions raised, then experiments
in pots are a valid approach, but we must further question what methods are
required to address these questions.
The principal reason for using the 15N-isotope dilution method is to distinguish
between nitrogen derived from the atmosphere and nitrogen derived from the soil.
If experiments are conducted in pots, the supply of nitrogen from the soil can be
carefully controlled, so there is little advantage in using the 15N-isotope dilution
method compared with simpler nitrogen balance methods.
The major concern with such studies under controlled conditions in pots is
that there is no guarantee that there is any correlation whatsoever between results
found under controlled conditions in the glasshouse and the response of the same
treatments in the field. This is largely due to the strong interactions between
nodulation and nitrogen fixation and a wide range of environmental factors, which
are described above.
268 K.E. Giller

13.5 Estimating Nitrogen Fixation in Field Settings

It can be argued that 15N-isotope dilution using labelled fertilizers has no

role to play in estimation of nitrogen fixation by trees, for the following
reasons.
• A wide variability in rooting patterns can be found even within a single
provenance of a tree species, as soil chemical and physical properties
exert a very strong influence on root distributions. As mentioned
before it is difficult to ensure that 15N is incorporated to depth and
unlikely that non-fixing plants can be identified that have matching
rooting patterns.
• One of the major advantages of legume trees is that they can be deep-
rooting, but this raises obvious problems in uniform 15N-labelling of
soil with depth. These are problems that also apply to measurement
of nitrogen fixation in herbaceous legumes but are amplified in
agroforestry systems because of the larger size and potentially deeper-
rooting systems of the trees.
• 15N-isotopes are expensive. Although it can be argued that the cost of

the isotopes is relatively small compared with other costs of field

experimentation and mass spectrometric analysis of samples, the wide
planting arrangements of trees in agroforestry systems demand the
use of large 15N-labelled microplots. There is, therefore, a strong
temptation to try to cut costs by not including border rows in the 15N-
labelled microplots, but sampling the whole area to which 15N-labelled
fertilizers have been applied. Obviously, this breaks the basic rules of
experimentation with 15N-labelling (see, for example, Stumpe et al.,
1989). Given the extensive rooting systems which have often been
observed in agroforestry trees, border rows are particularly important
(see Section 3.2).
This means that the 15N natural abundance method is probably the
only sensible choice for measuring nitrogen fixation by trees in the field,
but again problems should not be underestimated. The δ15N of soil
nitrogen may vary markedly with depth, although the extent to which this
is a problem is debatable (Boddey et al., 2000). Some studies have found
little change of δ15N in the plant-available nitrogen with depth, but there
is considerable variation between sites so this should be investigated if
possible. It is unlikely that a reference tree can be identified which will
have an identical pattern of root activity with depth, or uptake with time,
as that of the nitrogen-fixing tree under investigation. One approach to
this problem is to use a range of potential reference plants. This was
recommended for measurements of nitrogen fixation in grain legumes
(Boddey et al., 1990) but is especially applicable for nitrogen-fixing trees.
If most of the reference plants give δ15N values close to the δ15N of the total
Biological Nitrogen Fixation 269

Kjeldahl nitrogen of the soil on which they are growing, but one reference
plant consistently gives δ15N values significantly different from the rest,
then that is a reasonable indication that it is an aberrant reference value
and may be discarded. Such a situation was found when measuring
nitrogen fixation in Faidherbia albida in Malawi where mango (Mangifera
indica) consistently gave small δ15N values (2.7–4.8‰) compared with the
soils (6.4–11.5‰) and other non-fixing trees on the same sites (6.0–10.0‰)
(Phombeya, 1999).
The reason(s) why certain plants tend to become depleted or enriched
in 15N relative to their sources of nitrogen are not fully understood but
appear to be related to mycorrhizal colonization, drought and nitrogen
deficiency (Högberg and Alexander, 1995; Handley et al., 1996, 1999).
Högberg (1990) showed that ectomycorrhizal non-nodulating legume
trees (Brachystegia and Julbernardia spp.) in miombo woodland of Tanzania
were more depleted in 15N than arbuscular mycorrhizal-nodulating or
non-nodulating legume trees. Subsequent studies in Cameroon did not
confirm that these differences were due to the type of mycorrhizal infection
(Högberg and Alexander, 1995). Other research suggests that δ15N
depletion in trees may be related to fractionation during transfer from
mycorrhizas and depletion of δ15N in tree shoots may indeed be indicative
of mycorrhizal status (Hobbie et al., 1999, 2000). Problems of this kind
must be borne in mind if the δ15N method is to be used. As a general rule,
other indicators of nitrogen fixation such as nitrogen accumulation and
the ability of the tree to nodulate should be ascertained before this method
is used. Application of the 15N natural abundance method for measuring
nitrogen fixation by trees is reviewed in detail by Boddey et al. (2000).

13.6 Methods Based on Nitrogen Fixation Transport Products

Nitrogen is taken up from soil principally in the form of nitrate, whereas

fixed nitrogen is assimilated into amides or ureides depending on the host
legume before transport to the shoots. The proportion of nitrogen derived
from nitrogen fixation at any given time can be estimated based on the
relative proportion of the nitrogen in the xylem sap present as ureides
(Peoples et al., 1989). Samples of bleeding sap from cut shoots are taken
for analysis of total nitrogen and ureide nitrogen. This method can also
be applied by extraction of dried petioles and determination of the ureide
content, which makes sampling easier (Peoples et al., 1989).
Ureide production appears to be limited to legumes of the tribes
Phaseoleae, Indigoferae and Desmodieae (Giller, 2001), so that the only
agroforestry species confirmed to transport ureides are Cordariocalyx
gyroides and Desmodium rensonii (Herridge et al., 1996). Flemingia macrophylla
belongs to the tribe Phaseoleae and may therefore transport ureides (Giller,
270 K.E. Giller

2001), but this has yet to be confirmed. Comparisons of field estimates of

nitrogen fixation in Cordariocalyx with the 15N natural abundance method
showed close correspondence with estimates made using the ureide
method (Peoples et al., 1996). Reports of ureide production in species
within the tribe Robineae (such as Sesbania and Gliricidia) and the Mimoseae
are almost certainly due to artefacts produced by coloured compounds
during the analysis procedure. Attempts have been made to estimate
nitrogen fixation in amide-exporting legumes using xylem sap analysis,
but with less success (Peoples et al., 1989).

13.7 Estimating Total Amounts of Nitrogen Fixation

With all of the above methods, biomass estimates and tissue nitrogen
analyses are required to calculate actual amounts of nitrogen fixation,
using these estimates of %N from nitrogen fixation. Although it can be
seen that estimation of nitrogen fixation is difficult, there is still remarkably
little quantitative information given the potential importance of nitrogen
fixation in the long-term sustainability of agroforestry systems.