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II. Methods of Assessing N2 Fixation

II. Methods of Assessing N2 Fixation

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159



NITROGEN FIXATION BY TROPICAL LEGUMES

N



A



.n



FIG. 1. Diagrammatic representation of the principles involved in "N techniques for

measuring N2 fixation. The utilization of atmospheric N2 results in a lower "N composition in

the legume than measured in the nonfixing reference plant and is represented as a reduced

area in the "N sector of the leguminous plant. After Peoples er a / . (1988).



1. The reference plant lacks the ability to fix N2 and the l5N/I4N ratio

measured in its products of growth is the same as plant-available soil

mineral N.

2. The legume and non-N2-fixing reference plant explore a soil N pool of

identical l5N/I4Nabundance.



If a suitably precise mass spectrometer is available, the very small

differences in "N abundance between atmospheric N2 and soil (e.g.,

0.368-0.373 atom% "N in soil mineral N), which occurs naturally in many

agricultural soils, can be utilized for N2 fixation measurements (Shearer

and Kohl, 1986). More usually, the differences between N2 and soil N are

extended by incorporation of "N-enriched compounds into the soil (e.g.,

of between 5 and 95 atom% "N; Chalk, 1985). Alternatively, although less

frequently, differences between I5N composition of air and soil may also

be widened by applying "N-depleted (i.e., depleted in "N content relative

to atmospheric N2) materials to soil (e.g., 0.0016 atom% I5N ammonium

sulfate; van Kessel and Roskoski, 1988).



160



MARK B. PEOPLES AND DAVID F. HERRIDGE



1 . "N Enrichment



The use of methods involving the artificial adjustment of soil "N concentrations to measure N2 fixation has been extensively reviewed (e.g.,

Chalk, 1985; Hauck and Weaver, 1986; Danso, 1988; Ledgard and Peoples, 1988; Witty et al., 1988; Peoples e t a l . , 1989a). A major assumption is

that the legume and reference plant or crop absorb the same relative

amounts of N from the added "N and from the soil. The high cost of

instrumentation to measure 15N and expense of "N materials (U.S.$lO/g

of ["Nlnitrate of 10% enrichment) are limitations to the widespread use of

this potentially very accurate method. The main advantage of the technique is that it provides a "time-averaged'' estimate of P, which is the

integral of any changes in plant reliance on N2 fixation that may have

occurred during the measurement period. The calculation of P is independent of yield, although it is necessary to measure dry matter and N yield to

determine the amount of N2 fixed. Estimates of P are calculated from the

following:

P = l -



(atom% 15N excess legume N)

(utom% "N excess soil-derived N)



where atom% "N excess = (atom% "N sample) - (atom% "N air N2),

and atom% "N of air N2 = 0.3663. It can be seen that Eq. (2) is related

to (1).

The atom% "N of soil-derived N is generally estimated from the "N

enrichment of non-N2-fixing reference plants grown in the same soil over

the same period as the legume. It may be possible under certain conditions

to determine it directly from measurements on the soil mineral N (Chalk et

al., 1983).The choice of an appropriate non-N2-fixing reference plant is the

single most important factor affecting the accuracy of estimates of P.

Figures 2a-c illustrate three possible legume-reference plant relationships that are likely to occur in field experiments using "N enrichment.

When "N-labeled materials are applied to soil (most commonly in the form

of soluble inorganic fertilizer), a highly enriched layer is usually created

near the surface. Enrichment of soil mineral N generally declines rapidly

with depth. In Fig. 2a the legume roots have explored deep into the region

not artificially enriched with 15N,whereas roots of the reference plant have

been restricted to the uppermost enriched zone. The uptake of any soil N

by the legume from the unenriched region will, when the lsN/I4N compositions of the legume and reference are compared, lead to an overestimation

of NZfixation.

In the reverse situation (Fig. 2b), where roots of the reference plant have

grown below the "N-enriched zone, uptake of N from the unenriched soil



NITROGEN FIXATION BY TROPICAL LEGUMES



161



zone of

I5N enrichment



\~.............

..............

................

.................

..........................



*dd



..



decreasing w i t h

depth



~~~~



...............

.................



...

....................

........................



I5N enriched



.........................

..............................

..............................



-



n -.

atu

a l 15N

..

-.r -.

..



abundance



(:::::I



:fl.y&@: :::::::::::::::::::



....

....................

....

.'qy.V .....................

..............................



FIG.2. Diagrammatic representation of four situations that can occur during "N experimentation in the field. Figures (a) to (c) illustrate differences in the root growth of a nodulated

legume and a non-N2-fixingreference plant in hypothetical I5N enrichment studies. Application of "N-enriched material to the soil surface has created a zone where soil N is artificially

enriched with 15N. However, the "N enrichment decreases down the soil profile until natural

abundance levels are reached at depth. The fourth case study (d) illustrates a "N natural

abundance study where the 15N composition of plant-available soil N is relatively uniform

down the soil profile. After Peoples et a / . (1989a).



will result in a lowered "N content of the reference material and will lead

to an underestimation of fixation by the legume. It is in the ideal situation

(Fig. 2c), where roots from both test and reference plants explore a similar

volume of soil of similar "N enrichment, that accurate determinations of

symbiotic N2 fixation can be expected. However, even under these conditions, further errors can arise if there are substantial changes over time in

isotopic composition in the soil N pool combined with differences in the

patterns of N uptake by the legume and reference plants. A decline in soil



162



MARK B. PEOPLES AND DAVID F. HERRIDGE



lsN/14Nratio with time results from a loss of plant-available "N due to

uptake, leaching, or immobilization and the continuing release of I4N

compounds via mineralization of soil organic matter. Together, these

factors can lead to estimates of fixation that vary with choice of reference

species. Immobilized forms of "N have been used to ensure a more

gradual release of labeled N and to provide a more stable enrichment of

soil. These include the use of "N-labeled plant material, slow-release "N

formulations (e.g., "N incorporated into gypsum pellets), or the application of soluble "N salts with a readily available carbon source (e.g.,

sucrose) so that the "N is bound in the soil biomass (Boddey et al., 1984;

Giller and Witty, 1987). Errors associated with mismatching of the reference and N2-fixing legume are most important when P is small.

2. Natural "N Abundance



Almost all N transformations in soil result in isotropic fractionation. The

net effect is often a small increase in the I5Nabundance of soil N compared

with atmospheric N2 (Shearer and Kohl, 1986). In looking at such small

differences in I5N concentration, data are commonly expressed in terms of

parts per thousand (6"N or 0/00):

6"N



=



1000 x



(atom% "N sample) - (atom% "N standard)

(atom% "N standard)



(3)



where the standard is usually atmospheric N2 (0.3663 atom%). By definition, the 6"N of air Nz is zero.

The natural abundance method gives an integrated estimate of P over

time as with "N enrichment studies, but it can be applied to established

experiments (provided nonfixing reference material is available) because

no pretreatment, that is, "N application, is necessary. An estimate of P is

obtained by using the equation (analogous to (1) and (2))

P =



(6"N soil N) - (6"N legume N)

(6I5Nsoil N) - B



(4)



The 6"N of soil N is commonly obtained from a non-N2-fixing reference

plant. Similar estimates of P may also be obtained using direct soil measurements (Bergersen et a / . , 1989). The value B is a measure of isotopic

fractionation during N2 fixation and is determined by analysis of the 6"N

of total plant N accumulated by a nodulated legume grown in N-free

media.

Isotopic fractionation during N2 fixation is minimal but not zero and

should be taken into account. The value of B should ideally be prepared for



NITROGEN FIXATION BY TROPICAL LEGUMES



163



each new legume species studied (Peoples et al., 1989a). Some species

characteristically exhibit slight enrichment or depletion of 615N in various

plant parts when grown with N2 as the sole source of N (see Yoneyama et

al., 1984; Bergersen et al., 1988; Kohl et al., 1989). Critics of the natural

abundance method frequently cite the enrichment of I5N in nodules as a

potential source of difficulty when quantifying N2 fixation. The use of the

appropriate B value (e.g., in soybean it is - 1.30 O/OO when analyzing only

shoots, or a value of -0.79 if whole plants [shoot plus roots] are harvested;

Bergersen et al., 1988)when calculating P minimizes possible errors. Even

so, field results have indicated that well-nodulated roots make little contribution to total crop N and therefore have little effect on estimates of P

(Bergersen et al., 1989). There appears to be no evidence for significant

rhizobial strain-induced changes in 615N enrichment or depletion of different plant organs or whole-plant B values in tropical legumes (M. B. Peoples, unpublished data). Nonetheless, there can be dynamic changes in

615N of plant parts during organ development, so estimates of P should be

based on 615N of whole plants or total shoot N and not of single leaves or

individual plant parts (Bergersen et al., 1988).

Although the principles of the natural abundance technique are the same

as those underlying I5N enrichment, the main limitations are quite different (reviewed by Mariotti et al., 1983; Shearer and Kohl, 1986; Bergersen,

1988b; Ledgard and Peoples, 1988; Peoples et al., 1989a).An isotope ratio

mass spectrometer capable of accurately measuring differences of 0.1 O/OO

(about 0.00004 atom% "N) is required and sample collection and preparation require great care to:

1. Avoid contamination with "N-enriched materials.

2. Prepare uniform dry matter samples to avoid variation due to tissue

differences in "N abundance.

3. Avoid losses of minute quantities of nitrogen during Kjeldahl digestion and distillation, or during concentration of distillates before analysis

on the mass spectrometer. Such losses can change the "N abundance and

lead to poor replication of samples.



The accuracy of the technique will ultimately depend on the levels of

natural "N abundance of the soil. Low and/or variable soil 6"N values

will be unsuitable for assessing N2 fixation. A range of levels of 615N has

been measured in the mineral N component of tropical and subtropical

soils (Table 11; see also Ruschel et al., 1982). Fortunately, the natural "N

abundance of plant-available N is often sufficiently high and uniform for

reliable N2 fixation measurements in arable agricultural systems where

soils are regularly cultivated (Table 11). Although a gradual change in the

6"N of mineral N has been reported with soil depth for some rice-growing



164



MARK B. PEOPLES AND DAVID F. HERRIDGE



Table I1

Range of Levels of Natural Abundance of "N in Plant Available Soil N from Tropical and

Subtropical Soils

Location

Australia

New South Wales

Queensland

Western Australia

India

Indonesia

Java

Sumatra

Malaysia

Sungei Buloh

Taiping

Kuala Terengganu

Philippines

Luzon

Leyte

Thailand

Chiang Mai

Mae Taeng



Site use



I5N natural

abundance (O/OO)



SE"



Reference"



0.4

0.2



I

2

3

4

5



Cropping

Cropping

Pasture/cropping

Cropping

Cropping



8.7- 13.9"

6.6

2.6-14.8"

6.3

4.0-5.2"



Tree legume stand

Cropping

Tree legume stand



2.0

6.0

2.5-4.2'



Rubber plantation

Cropping

Rubber plantation



3.2-3.6

10.1-16.6

1.2-4.3'



Cropping

Tree legume stand



5.1 - 13. 5 c

1.0-3.8"



0.7



Cropping

Cropping

Cropping



8.0

3.9

4.5-8.4'



0.2

0.5



0.7

0.2



6



7

0.8

8

9

10



11

12

12



Standard error of the estimate mean at one site.

I . Herridge et al. (1990); 2. M. B. Peoples and M. Bell (unpublished data); 3. H. V. A.

Bushby (unpublished data); 4. Ofori et ul. (1987); 5. Yoneyama et a/. (1990); 6. D. P.

Nurhayati, T. Ibrahim, and M. B. Peoples (unpublished data); 7. Norhayati er al. (1988);

8. A. W. Faizah and M. B. Peoples (unpublished data); 9. Watanabe er al. (1987);

10. A. S. Almendras, P. J. Dart, and M. B. Peoples (unpublished data); I I . Rerkasem e t a / .

(1988); 12. A. Bhromsiri, C. Sampet, and M. B. Peoples (unpublished data).

Range of values detected at different field sites.

a



soils (Watanabe et al., 1987) (possibly a result of repeated, heavy applications of N fertilizers), analysis of many other soils suggests that this may

not be a general phenomenon. More often the level of "N natural abundance of plant-available N is relatively constant with soil depth (as depicted in Fig. 2d; Watanabe er al., 1987; Bergersen, 1988b; G. L. Turner,

M. B. Peoples, and F. J. Bergersen, unpublished data) and does not appear

to change rapidly with time (see reference plant and soil data in Ofori er al.,

1987; Norhayati et al., 1988; Bergersen et al., 1989; Herridge et al., 1990).

Therefore, the major limitation of 'N enrichment techniques (i.e., choice



NITROGEN FIXATION BY TROPICAL LEGUMES



165



of appropriate reference plant) is likely to be less critical with the natural

abundance method. Certainly, field studies have shown that the use of

different reference plants does not greatly influence the calculation of P

(Rerkasem et al., 1988; Bergersen et al., 1989; Yoneyama et al., 1990).

Where natural abundance and "N enrichment techniques have been compared, field estimates of N2 fixation were similar, with similar precision

(see Ofori et al., 1987; Ledgard and Peoples, 1988; Norhayati etal., 1988).



B. N-DIFFERENCE

METHOD

The simplest estimates of N2 fixation reported in the literature were

obtained by measuring the total amount of N accumulated by legume

crops. Such determinations are based on the arbitrary assumption that the

legumes derive all of their N from N2 fixation. Values obtained will almost

certainly overestimate N2 fixation. An adaptation of this N yield technique

has been used to assess N2 fixation by tree species, where increments of

soil N under trees and N contained in the plant biomass have been measured and summed (Dommergues, 1982). Results obtained using such

a method should also be treated with caution since the observed increments

cannot be attributed solely to symbiotic N2 fixation. Other processes, such

as extraction of N by roots from deep soil horizons or from the water table,

could contribute to the accumulation of N.

A true measure of N2 fixation based on legume N yield can only be

obtained when the contribution of soil N to total plant N is determined.

This is often estimated by growing a non-N2-fixing crop in the same soil

under identical conditions as the legume, usually in adjacent plots. The

difference in total N accumulated by the legume (NI) and nonfixing control

(Nnf) crops is regarded as the contribution of N2 fixation to legume

growth. Thus, N2 fixation is calculated as

N2 fixed = NI



-



Nnf



(5)



Using the crop N data for the unfertilized plots from Table I as an

example, N2 fixed by the nodulated soybean is calculated to be 128 Kg

N/ha using the nonnodulating soybean as the nonfixing control and 150 kg

N/ha using the nonlegume Ragi. These calculations will be dependent on

the accuracy of measurements of crop dry matter and N content, which in

turn will largely be determined by errors associated with sampling and

subsampling (Hunt et al., 1987).

There are two basic assumptions inherent in the use of the N-difference

procedure:



166



MARK B. PEOPLES AND DAVID F. HERRIDGE



1 . The N contained in the non-N2-fixingcontrol plants is derived only

from soil N.

2. The legume and control crops assimilate the same amount of soil N.



The N-difference method is a relatively simple procedure and can be

used when facilities for only total N analysis are available. However,

because of the underlying requirement that the legume and control plants

utilize equivalent amounts of soil N, the choice of control crop is of utmost

importance. Ideally the two plant types should explore the same rooting

volume, have the same ability to extract and utilize soil mineral N, and

accumulate soil N over the same period of time. A non-N2-fixing control

plant may be ( a ) a non-legume; (6) an uninoculated legume (requires the

soil to be free of effective Rhizobium spp.); or (c) a nonnodulating legume,

preferably an isoline of the test legume.

Unfortunately, there are often substantial differences between fixing

and nonfixing plants in their capacities to use soil N. Even when a supposed “ideal” control plant is used (e.g., a nonnodulating isoline of the test

legume), errors in calculating N2 fixation may result because of differences

in root morphologies (Boddey et al., 1984). Residual levels of soil mineral

N may also be higher following a legume crop than a nonfixing crop

(Herridge and Bergersen, 1988). Such observations led Evans and Taylor

(1987) to propose a modification of the N-difference procedure to improve

accuracy when legume and control are not well matched. In this method

the amount of mineral N in the soil under the legume (Soil (1)) and nonfixing

(Soil (nf)) crops is measured at the completion of crop growth and the

difference between the two added to the difference in crop N yields. Thus

Eq. ( 5 ) becomes

N2 fixed = [NI - Nnf]



+ [Soil (I)



-



Soil (nf)]



(6)



The importance of choosing a particular control plant depends ultimately on the level of plant-available soil N and the amount of N2 fixed. If

the control plants accumulate very much less N than the legume, differences in uptake of soil N between the two plant types will not greatly

influence the determination of N2 fixation.

Comparisons of N2 fixation calculated by N-difference with estimates by

”N dilution have often shown good agreement in soils low in N or where

recovery of ”N label is equal in the legume and control (Chalk, 1985).

Under such conditions, however, agreement of estimates is mathematically inevitable and in itself does not constitute an independent

confirmation that either technique is correct (Urquiaga and Boddey,

1987).



NITROGEN FIXATION BY TROPICAL LEGUMES



167



C. UREIDEMETHOD

Xylem sap carries N-containing compounds from the roots to the shoots

of field-grown legumes originating from ( a ) nodules as assimilation products of N2 fixation and (b) soil mineral N taken up by the roots.

The first stable product of N2 fixation in the legume nodule is ammonia.

The ammonia is released from the bacteroids to be assimilated into glutamine and glutamate in the infected host cell via the coupled activity of the

enzymes glutamine synthetase (GS) and gluatmate synthase (GOGAT).

Table I11

Nodulated Legumes That Transport Ureides as a

Major Nitrogenous Component of Xylem Sap"

Genus

Albizia

Cajanus

Calopogonium

Cenirosema

Codariocalyx

Cyamopsis

Desmodium

Glycine

Hardenbergia

Kersiingiella

Lablab

Macroptilium

Macrotyloma

Phaseolus

Psophocarpus

Pueraria

Tedehegi

Vigna



Voandzeia



Speciesh

lophaniha

cajan

caeruleum

pubescens

gyroides

reiragonoloba

discolorlrensoniiluncinaium



max

geocarpa

purpureus

atropurpureum

ungorum

lunatusluulgaris

ietragonolobus

jauanicalphaseoloides

triqueirum

angularislmungolradiatal



trilobalunguiculatal

umbellaia

subierranea



Forty percent or more of total xylem sap N is

estimated to be in the form of allantoin and allantoic

acid.

Adapted from Pate and Atkins (1983);Schubert

(1986); Peoples ei al. (1989a), and includes unpublished information from D. F. Herridge, B. Palmer,

M. B. Peoples, F. D. Dakora, C. A. Atkins, and

J. S. Pate.



168



MARK B. PEOPLES AND DAVID F. HERRIDGE



Despite the production of glutamine as the initial product of ammonia

assimilation, it is rarely the major N solute transported in the xylem of

nodulated plants (Pate and Atkins, 1983; Peoples et al., 1987). Secondary

reactions involving the transfer of the amide- or amino-N of glutamine to

other products comprise important metabolic processes within the functional nodule (Pate and Atkins, 1983; Schubert, 1986). In many nodulated

legumes of tropical origin, the ureides, allantoin and allantoic acid, are the

predominant N compounds exported from the nodule and transported in

the xylem (Table 111; e.g., soybean, see Fig. 3).

Nitrate and ammonium ions are the two forms of mineral N taken up by

plant roots. In most agricultural soils, where nitrification takes place rapidly, nitrate is considered to be the dominant N source for plant growth

(Stevenson, 1982). Solutes derived from soil mineral N under these conditions will be transported in the xylem as free nitrate or as organic products

of nitrate reduction. Characteristically, nitrate reduction assumes a minor

role in assimilating nitrate in the roots of most tropical and subtropical

legume species (Andrews, 1986; Wallace, 1986), and, as a consequence,

much of the incoming nitrate is transported in an unreduced form (Fig. 4).



R



0001



-1



- N



+ NO3



FIG.3. Composition of N solutes of xylem sap collected from fully symbiotic (nodulated

plants maintained on N-free complete nutrients) and nonnodulated (fed zero or 10 mM

nitrate) soybean plants. Saps were collected as bleeding sap from decapitated roots or from

detached nodules. Asn = asparagine; Gln = glutamine. After Peoples and Gibson (1989).



169



NITROGEN FIXATION BY TROPICAL LEGUMES



The portion of the nitrate that is metabolized will be reduced to ammonia

by the combined action of root nitrate reductase and nitrite reductase (Pate

and Atkins, 1983). The ammonia will then be assimilated by GS and

GOGAT as in the nodule, though, unlike the nodule, ureides do not play a

major role in the subsequent metabolism or transport of this N from the

roots. Rather it is the amide, asparagine, that is the exported end product

of nitrate reduction (Fig. 3). The same appears to be true if ammonium

rather than nitrate is taken up by roots. The incoming ammonium ions are

incorporated into amides and amino acids rather than ureides (Peoples et

al., 1989b). Therefore, in the absence of nodules, ureides represent only a

very minor component of the total N of xylem sap (Fig. 3).

Because there are substantial differences in the principal forms of N

transported in the xylem between highly symbiotic and unnodulated or

poorly fixing legume plants, it is possible to use the abundance of ureides

relative to the other N components in xylem sap as an indirect 'measure of

the proportion (P)of plant N derived from N2 fixation (Fig. 4). Glasshouse

calibration experiments (McClure et al., 1980; Pate et al., 1980; Rerkasem

et al., 1988; Peoples et al., 1989b; Herridge and Peoples, 1990) indicate

that there is a predictable progressive decrease in xylem ureides and a

compensatory increase in amino acid and nitrate contents as N2 fixation

PARTIALLY SYMBIOTIC



FULLY SYMBIOTIC



N,



N,



SHOOT AXIS



1 ly



+ NO;



NON- SYMBIOTIC

NO;



A



NoDwM

1



4' 1

a



RooT



N2T

-w

NO;



b



XYLEM TRANSPORT OF:



a



C



UREIDE I AMINO COMPOUNDS

NITRATE 6 AMINO COMPOUNDS



FIG.4. Pathways of N transport from the root systems of ureide-exportinglegume species

when growing in (a) nitrate-free soil, (b) moderate levels of soil nitrate, and (c) high levels of

soil nitrate.



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II. Methods of Assessing N2 Fixation

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