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II. Methods of Estimating Fixation by Crops

II. Methods of Estimating Fixation by Crops

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Experiments on legumes growing on artificially impoverished soils are not

uncommon in the literature. The substrate used by Lyon and Bizzell was an

artificial one and for that reason the data they obtained should not be extrapolated

to estimate fixation by farm crops.

Long-Term Nitrogen Balance Studies Using Lysimeters

A lysimeter is an enclosed soil system in which the addition and removal of

nutrients, water, and plant material can be controlled and measured. Although

many designs have been described (Kohnke et al., 1940), the basic construction

consists of a tank or box placed in the ground and filled with soil. Leachate from

the soil in the tank is collected from the bottom of the lysimeter. Lysimeters have

been used in long-term studies on nitrogen balance in crops under different

management systems (Chapman et al., 1949). In addition, lysimeters can be

used to compare and calibrate different techniques for estimating N fixation

under controlled conditions (Williams et al., 1977).

Several disadvantages are inherent in lysimeter studies. They are expensive to

install because they require the excavation of large volumes of soil and must be

constructed of materials resistant to corrosion. Due to their expense, the size and

number of lysimeters, and thus the number of treatments and replications, is


In some lysimeter studies, long-term nitrogen balances have shown unaccounted losses of nitrogen (Collison et al., 1933). This is attributed to volatilization of N from the lysimeters (Chapman et al., 1949; Patwary and Raikavich,

1979), although this assumption has been challenged (Craswell and Martin,




An adjusted measure of fixation by the nitrogen accumulation technique is

obtained when the contribution of soil N to the total N of legumes is estimated.

This correction for the contribution of soil N is obtained by growing a nonfixing

plant in comparison with the N-fixing legume. Total N content of the nonfixing

crop (derived solely from soil N) is subtracted from the total N content of the

N-fixing legume. The difference between the values is assumed to be the quantity

of N derived by N fixation. This procedure is often referred to as the “difference” method (Williams et al., 1977). Three versions of the difference

method are commonly used.

1 . Comparison of a Legume with a Nonlegume

Soil N contribution to a fixing legume is estimated by growing a nonlegume

concurrently with the legume. Annual small grains such as wheat and oats have



been used (Rizk, 1966). In studies of forage legumes, however, perennial grass

species are used. This is preferred in studying N fixation in grass-legume mixed

pastures. Sprague (1936) used wheat as a control crop to estimate the soil N

contribution. The wheat contained 38 kg N/ha and the vetch 149 kg N/ha. By

subtraction, he estimated the net fixation of the vetch to be 111 kg N/ha.

Using a nonlegume as a control in the difference method requires two major

assumptions. First, the nitrogen contained in the nonlegume is assumed to arise

solely from the soil N pool. Secondly, the legume and nonlegume will take up

soil N in proportion to the amount available, and differences due to growth

patterns and root morphology will not be significant. The validity of the second

assumption is open to question. No evidence exists that shows the exploitation of

soil N is equal between a legume and its nonlegume control.

The fixation estimated will obviously depend on the arbitrary choice of nonlegume control. Wagner (1954) calculated that the N fixation of ladino clover

was 165 kg/ha when orchard grass was used as a control or 189 kg/ha when tall

fescue was the control.

2 . Comparison of a Legume with a Nonnodulating Legume

Another approach to the difference method utilizes the existence of nonnodulating legume genotypes. Soybean genotypes with nonnodulating characters

have been described (Williams and Lynch, 1954). The nonnodulation character

is controlled by a single recessive gene, labeled rj, . This nonnodulating gene has

been backcrossed to produce near-isogenic lines. The advantages of using nonnodulating lines as a control species are that the growth pattern, root

morphology, and N uptake patterns are assumed to be nearly identical. However,

the nodulated isoline may absorb more soil N than the nonnodulated isoline

(Ruschel et al., 1979). This suggests a synergistic effect of fixed N on N derived

from other sources.

Weber (1966) used nonnodulating isolines of soybean to estimate N fixation

under different levels of applied fertilizer nitrogen. By subtracting the N content

of the nonnodulating isoline from that of the nodulated soybean, N fixation rates

of 14.7-84 kg/ha were calculated.

In soybeans, isolines are available for only a few cultivars. Unfortunately,

near isogenic nonnodulating and nodulating cultivars are not yet available for

other legume species.

3 . Comparison of Inoculated and llninoculated Legumes

Another difference method is a comparison of single cultivars grown on inoculated or uninoculated soil. A variation of this procedure uses separate plots

inoculated with effective or ineffective strains of rhizobium (Nutman, 1973). In

this situation, the identical variety is used for the control and treatment, with the



only variable being the presence or absence of nodules (or effective and ineffective nodules). This method requires a soil free of native rhizobium species

capable of establishing an effective symbiosis with the legume being studied.

Bezdicek et al. (1978) compared inoculated and uninoculated soybeans under

field conditions. Uninoculated soybeans accumulated 76 and 105 kg N/ha in the

2 years, while nodulated soybeans yielded 387 and 368 kg N/ha. By difference,

the net fixation was calculated as 31 1 and 263 kg N/ha. In areas where legumes

are routinely grown, soils lacking the appropriate rhizobium species may not be

available. When using this technique, considerable care is required to prevent

contamination of the uninoculated soils with rhizobium. For these reasons this

procedure has limited application in the routine evaluation of N fixation.

Using the difference method with nonnodulating legumes or uninoculated

plots requires the assumption that the root structure and function are not altered

by the presence of nodules. This assumption has yet to be proven experimentally.

4 . N Fertilizer Equivalence

In some studies, an indirect estimate of nitrogen contribution from a legume to

a nonlegume is used to estimate N fixation. Forage legumes and grasses are often

grown together in pasture systems. The legumes have been observed to stimulate

the productivity of the grass species (Lyon and Bizzell, 1911). If the response of

the grass in the mixed stand is compared to the response of the grass in a pure

stand with different amounts of applied fertilizer N, then an estimate of the N

contributed to the grass by the legume is obtained.

This method has been used in the studies of pasture management systems.

Cowling (1961) calculated the “indirect effect” of clover on grass as the amount

of fertilizer N applied to a grass plot to give a yield equivalent to the grass

component of a clover-grass mix. He found that the effect of clover on cocksfoot

was equivalent to 117-134 kg fertilizer N/ha.

Another variation of the nitrogen contribution technique is to estimate the

value of a legume to a following crop. Green manure crops have often been used

to increase soil N (Kroontje and Kehr, 1956; Kamprath et a l . , 1958; Sprague,

1936). The N added to the soil by the legume crop gives a yield in the successive

crop that is compared to the response to different levels of added N fertilizer.

Kroontje and Kehr (1956) estimated that vetch added the equivalent of 107 kg

N/ha to the soil. This resulted in a significant increase in barley yields following

the vetch crop. Similarly, Sprague (1936) estimated that a winter crop of vetch

was equivalent to 671 kg NaNO,/ha.

In comparing the response of follow crops, urea can be used as the nitrogen

fertilizer (de Souza, 1969) because it contains no other nutritional element that

might contribute an effect. A N fertilizer is subject to leaching and volatilization,

whereas the decomposing forage crop releases N more slowly and efficiently to



the nonlegume crop. Therefore the “equivalents N ” is likely an overestimate of

N fixation. The beneficial effects of legumes on nonlegumes are not a direct

measure of N fixation. However, they do show the utility of green manures in

adding N to the soil, and the cost of the fertilizer spared may serve as an

indication of the economic value of N fixation.


The use of 15N isotopes for studying N uptake by plants was recently reviewed

here (Hauck and Bremner, 1976). Very few of the articles reviewed related to the

use of I5N for estimating fixation by legume crops. The technique is likely to

become more common, however, because of the lowered cost of the isotope and

improvements in methodology. These include relatively inexpensive mass spectrometers designed for low-mass compounds (e.g., Micromass) and increased

precision by using double introduction and double collection.

Because it is a direct method, fixation of I5N2 remains the method of choice for

checking the validity of other estimates of fixation (Burris, 1972). The cost of

I5N2 and the volumes that would be required to test replicate plant samples over a

growing season preclude estimates based on that gas. However, the technique

has been used to test assays based on other methods. The methods are essentially

those developed by Burris and Wilson (1957; Bums, 1972). Typically, nodulated roots (Saito et al., 1980) or nodules (Hudd et al., 1980) are briefly incubated in a chamber with a gas phase enriched in 15N1. The fixation is stopped by

the addition of strong acid, the tissue is analyzed for total N (Kjeldahl) and I5N

by mass spectrometry (Smith et al., 1963) or emission spectrometry (LloydJones et al., 1977).

The major limitation to mass spectrometer methods is the high cost involved

for the instruments and the isotopes. The procedures are fraught with potential

errors. Although the proper techniques are well documented (Bremner, 1965), it

remains true that the method is demanding and requires skilled operators.

When N gas is excited by a high-frequency oscillator, the wavelengths of

emitted light depend on the isotope composition. Commercial emission spectrometers, less expensive than mass spectrometers, are available for measuring

15N. The instruments are not as precise as mass spectrometers. Their major

advantages are that they require only small samples (10 p g N) and they are not so

demanding of operators.

I . Methods Based on Isotope Dilution

Nitrogen fixation can be estimated by isotope dilution. In this method the

fixing crop and a nonfixing control are grown in soil to which I5N has been added



as a small amount of labeled nitrate or ammonium (McAuliffe et al., 1958; Legg

and Sloger, 1975). The N in the control plant should have the same 15Ncontent

as the available soil N. The plant obtaining part of its N from the atmosphere will

have less of the isotope. The percentage of total N from fixation is calculated as

%N=( 1-

at. % 15N excess test crop

at. % I5N excess control crop

) x 100

This technique involves the assumption that the isotope incorporated into soil

N is equally available to both crops. Moreover, if the soil N is low, the nonfixing

crop may grow less well than the test crop and thus not be a comparable control.

Fried and co-workers (1975, 1977) proposed the “ A N value” modification.

Adequate labeled fertilizer N can be added to the soil for the nonfixing control to

promote growth. I5N in fertilizer N is added to soil for test plants if the effect of

fertilizer N on fixation is required. The 15Ncontent of the plants is measured after

harvest. For each crop and treatment, the “AN” value is determined. The A N

value (Legg and Stanford, 1967) is an indication of how much of a nutrient is

available to a crop and is expressed as

AN = B ( l - y ) / y

where B is the amount of fertilizer N added (kilograms N per hectare) and y

is the proportion of N in plant derived from that added:

excess 15N in plant

= excess 15N in fertilizer

The fraction of legume N from fixation is calculated as

Nitrogen fixed (kg/ha) = (AN test plant - A N nonfixing control)

N in crop x excess 15N


N in fertilizer x excess I5N

It should be possible to calculate for each crop and treatment the contribution

made by soil, fertilizer, and symbiotic fixation. Procedural details and potential

sources of error in these methods have been reviewed by Hauck and Bremner

(1976) and Rennie et al. (1978).

3 . Methods Based on Natural Isotope Abundance

There are very slight isotope effects during chemical or biological processes

involving N compounds.

Symbiotic fixation seems to favor a very slight increase of 15N. This can be

determined by growing a plant on N-free nutrient so that all its N arises by

fixation. The isotope discrimination of fixation for soybean has been calculated

as 1.0038 (Bardin, 1977) and 1.0014 (Amarger et al., 1979) but also as 0.999

(Kohl and Shearer, 1980).



Denitrification produces N 2 lowered in 15N and the nitrate remaining in

soil slightly enriched. The degree of enrichment is measured as parts per thousand difference from the l5NP4N ratio in a standard (usually atmospheric

nitrogen) (Hauck and Bremner, 1976)

615N = (15NP4N) - (l5NP4N) atm

(I5NP4N) atm

where one 6


unit equals 0.00037 at. %



A plant obtaining all its N from soil (e.g., nonlegume or nonnodulated

control) will have a slightly enriched I5N relative to the atmosphere. A plant

obtaining N from symbiosis will have a lower I5N composition. The I5N enrichment is not uniform throughout the plant, although it is claimed that the seed

content is representative of the whole soybean plant (Shearer et al., 1980). The

percentage of N from fixation is calculated (Domenach et al., 1979):

% N fixed = 100 x

(% 15N control - % I5N legume)

% I5N control - (% 15N airlb)

where b is the isotope discrimination of fixation.

An appealing advantage of this method of estimating fixation is that it does not

necessitate the purchase of isotopic N to add to soil. The major disadvantage is

that the enrichment may not be uniform in the soil (Rennie et al., 1976).

Karamanos and Rennie (1980) found that the P 5 N dropped with depth in welldrained profiles but was constant to a depth of 5 m in upper slopes. Pedogenic

processes produced marked differences in the 6I5N profile within short distances.

These results extended and confirmed those of previous workers that there is an

unpredictable lack of uniformity of enriched N in soils.

The possible isotope discrimination due to symbiotic fixation may favor the

fixation of I4N. If this is not included in the calculation, the level of fixation will

appear to be higher than it is (Rennie et al., 1978).

Experienced critics (Hauck and Bremner, 1976) view the technique based on

natural abundance as giving only qualitative or semiquantitative information.

However the standard errors of the procedure when applied to field-grown

nodulating and nonnodulating soybeans compared favorably with the errors of

estimation based on N difference (Kohl et al., 1980).



Nitrogenase reduces acetylene to ethylene and, so far, it is the only biological

agent reported to do so. Typically, freshly excised roots are incubated in a

chamber with 1-20% C2H, for 30-120 min. A sample of the gas phase is then



removed and the ethylene produced is measured by gas chromatography. Innumerable variations of the method have been described (Hardy et al., 1973).

The principal assumption in the method involves the ratio of acetylene reduced

to nitrogen fixed. The reduction of nitrogen to ammonia uses six electrons, while

the production of ethylene requires two. Therefore, the ratio 3 : 1 was originally

assumed, i.e., a mole of ethylene was equivalent to % mole of N 2 reduced. It is

now realized that the reaction of nitrogenase approximates

N2 + 8 H+ + 8e ---t 2 NH,

+ H?

Protons and acetylene compete for electrons, and, with the amounts of GH,

usually used, only small amounts of H2 may appear. In the intact nodule the

hydrogen may be metabolized by hydrogenase in appropriate strains of rhizobia

and therefore not be detected.

There is no adequate method now for calibrating ethylene formation with

nitrogen fixation, but it seems that the ratio is approximately 4 : 1 for legumes

(Hardy et al., 1973).

The acetylene reduction method has the advantages of sensitivity, speed, and

economy. A detection limit of pmole C,H,/ml gas permits estimation of nitrogenase activity even when only a few nodules are forming. It is possible to

measure 40-80 samples/operator-day.

The operation of a gas chromatograph can be easily learned, and the analysis

may be conducted by semiskilled staff. No expensive reagents are necessary. If

sensitivity is not required, there are inexpensive alternatives to measuring

ethylene (LaRue and Kurz, 1975).

Because the plant is usually destroyed in the assay, acetylene reduction is a

one-time estimate of fixation. The incubation must be done immediately after the

root is harvested. There is a diurnal variation in fixation by many legumes

(Sloger et al., 1975). Much plant-to-plant variability is observed, and the entire

nodulated root may not be recovered from the soil. Therefore an estimate of

fixation over a growing season by a crop requires a mathematical summation of

many frequently obtained assays on replicate plots.



Several indirect methods have been used for estimating the nitrogen fixing

ability of a legume. These include indices of nodulation-number of nodules,

fresh or dry weight of nodules, and leghemoglobin concentration in nodule or

amount per plant. Within a single cultivar these may be closely related to nitrogen fixation and will continue to be useful in screening rhizobial strains or

scoring the effects of environment on nodulation. We have seen no evidence,

however, that these nodule-related characters can be used to calculate the amount



of fixation by crops. Nodules that are pink or red internally are only an indication, and not a proof, of nitrogen fixation (de Souza, 1969).

The concentration of the ureides allantoic acid and allantoin in the shoot is

positively correlated with the amount of fixation by soybeans grown in the

greenhouse under defined conditions (McClure er al., 1980). This suggests that

estimates of fixation might be made by assays, nondestructive to the plant, of N

compounds in the shoot parts. Several laboratories are pursuing this topic. While

there are indications that it may aid in the qualitative ranking of cultivars, the

procedure seems unlikely to measure the amount fixed by a crop.



Most published estimates of fixation by forages assume that plant N arises only

from N fixation. Excluding these, there remain very few estimates, most of

which are based on long-term lysimeter studies (Table I). Except where noted, all

studies used pure stands of legumes.

Chapman et al. (1949) studied the nitrogen balance of vetch (Vicia villosa)

and sweetclover (Melilotus alba) used as winter cover crops at Riverside,

California, over a 10-year period. Only one lysimeter per treatment was used, due

to their large size (3.04-m diameter) and small number (12). The legumes were

grown in a winter rotation and incorporated into the soil prior to planting a

summer crop of barley or sudan grass. Nitrogen fixation of the legumes was

estimated by the increase in soil N due to the incorporation of the green manure

into the soil. A 10-year average of N added to the soil gave an annual fixation

rate of 140 kg N/ha for sweetclover and 184 kg N/ha for vetch.

Estimates for alfalfa (Medicago sativa), white clover (Trifolium repens), red

clover (Trifolium pratense), and Korean lespedeza (kspedeza stipulacea) came

from an 1 1-year lysimeter study by Karraker et al. (1950) in Lexington, Kentucky. They studied the nitrogen balance in a continuous cropping system with

forage legumes and Kentucky bluegrass (Poa prarensis). The lysimeters were 57

cm in diameter, and each treatment was replicated twice.

Although the authors presented only the total N accumulation over the 1 1-year

experiment, the presence of the Kentucky bluegrass control permits our calculation of the average annual N fixation by the difference method. Averages for

each legume, minus the bluegrass control, are 212, 128, 154, and 193 kg N/ha

for alfalfa, white clover, red clover, and Korean lespedesa, respectively.

Jones et al. (1977) used a lysimeter-difference technique to estimate N fixa-

Table I

Estimates of Nitrogen Firation by Forage Crops

Contml species

Duration of

experiment (years)






Geneva. NY

Lexington, KY

R m m o u n t , MN

Lexington. KY

Northern Ireland

Beltsville, MD

Collison cr d.,1933

Karralicr cr al.. 1954

Heichel cr d.,I 9 8 1

Karraker cr ol.. 1950

Halliday and Pate, 1976

Wagner. 1954

Karraker er 01.. 1950

S p g u e . 1936

chapman cr al.. I949

Sprague. 1936

Phillip and Bennca. 1978

Joms er al.. 1977

Rizk. 1%2

165- 189



Isotope dilution-"A"


C& reduction



I 7b










"A" value



Winter wheat

Soh chess

Soh chess




Lexington, KY

New Brunswick. NJ

Riverside, CA

New Brunswick, NJ

Hopland, CA

Davis, CA

Giza,E g y p



Winter wheat


New Bmnswick. NJ

Sprague, 1936



Winter wheat


New Brunswick. NJ

Sprague. 1936




Winter wheat



Kentucky bluegrass


New Brunswick, NJ

Riverside, CA

Lexington. KY

Sprague. 1936

chapman e r a / . , 1949


ct d..1950


(Medicago sariva)




White clover

(Trifoliurn repms)

Ladin0 clover

(Trifoliurnrepens var. Ladino)

Red clover

(Medicago prmense)

Sweet clover

(Mclibrn a h )


(Trifoliurn subrerraneurn)

Egyptian clover

(Trifolium alexandrinurn)

Alsike clover

(Trifolium hybridurn)

Crimson clover

(Trifoliurn indica)


(Vicia vilbsa)

K o m kspedeza

(kspedew sripulacca)



aDuration given in months.

*Winter crop rotation.


I lob



Kentucky bluegrms

Reed canarygrass

Kentucky bluegrass




Orchard grass-call fescue


Kentucky bluegrass

Winter wheat








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II. Methods of Estimating Fixation by Crops

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