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Technical paper 2: Biological nitrogen fixation

K. Mulongoy


2.0 Performance objectives
2.1 Introduction
2.2 Mechanism of biological nitrogen fixation
2.3 Specificity and effectiveness
2.4 Factors limiting biological nitrogen fixation
2.5 Estimation of nitrogen fixation
2.6 How to increase BNF and N2 fixing ability
2.7 Summary
2.8 Feedback exercises (Find out answers from the text)
2.9 Suggested reading
2.10 References


2.0 Performance objectives

Technical Paper 2 is intended to enable you to:

1. Describe briefly the mechanism of biological nitrogen fixation.
2. Discuss edaphic, climatic and biotic factors limiting biological nitrogen fixation.
3. Describe two simple methods of BNF estimation.
4. Discuss four major approaches to enhance biological nitrogen fixation.

2.1 Introduction

Biological nitrogen fixation (BNF) is the process whereby atmospheric nitrogen (N=N) is reduced to ammonia in the presence of nitrogenase. Nitrogenase is a biological catalyst found naturally only in certain microorganisms such as the symbiotic Rhizobium and Frankia, or the free-living Azospirillum and Azotobacter.

Biological nitrogen fixation is brought about bath by free-living soil microorganisms and by symbiotic associations of microorganisms with higher plants. Our main interest in this paper centers on the legume-Rhizobium symbiosis. Leguminous plants fix atmospheric nitrogen by working symbiotically with special bacteria, rhizobia, which live in the root nodules. Rhizobia infect root hairs of the leguminous plants and produce the nodules. The nodules become the home for bacteria where they obtain energy from the host plant and take free nitrogen from the soil air and process it into combined nitrogen. In return, the plant receives the fixed N from nodules and produces food and forage protein.

2.2 Mechanism of biological nitrogen fixation

The biochemical mechanism of N2 fixation can be written in simplified form as follows: nitrogenase

The above mechanism indicates that N2-fixing systems can thrive in soils poor in N. that they are a source of proteins, and that they provide N for soil fertility. Adenosine triphosphate (ATP) is the source of energy necessary for the cleavage and reduction of N2 into ammonia. In rhizobia, for instance, ATP results from oxidative degradation of sugars and related molecules. These sugars are manufactured by the host-plant during photosynthesis and transferred to the nodules. In general, for each gram of N2 fixed by Rhizobium, the plant fixes 1-20 grams carbon (C) through photosynthesis. This is an indication that symbiotic N2 fixation requires additional energy which, in nitrate-fed plants, can be used to produce more photosynthates (products of photosynthesis). The extra energy cost of N2 fixation can, however safely be carried by most field-grown legumes with little or no loss of production.

It is usually accepted that N2 fixing systems require more Phosphorus (P) than non-N2-fixing systems. Phosphorus is needed for plant growth, nodule formation and development, and ATP synthesis, each process being vital for nitrogen fixation.

Nitrogen fixation, which involves the chemical reduction of N2 to NH3 or NH4, requires a source of electrons. Sources of electrons for the nitrogenase activity vary with the organism. They are all small proteins and highly reductive molecules such as flavodoxin, ferredoxin, nicotinamide, or ademine dinucleotide (phosphate).

Nitrogenase is an oxygen sensitive enzyme. The low oxygen tension condition is realized through compartmentation in cyanobacteria (heterokysts in Anabaena azollae), active respiration (in Azotobacter), synthesis of leghemoglobin (in Rhizobium legume). Leghemoglobin is a macromolecule synthesized by both symbiotic partners, the rhizobia and the host plant. Rhizobium synthesizes the heme portion, and the plant the globine. Like human hemoglobin, leghemoglobin fixes O2. It is responsible for the red or brown color of active (i.e., N2-fixing) nodules. Non-N2-fixing nodules have a white nodule content, or a green content when the globine has degenerated.

2.3 Specificity and effectiveness

There are roughly 1,300 leguminous plant species in the world. Of these, nearly 10% have been examined for nodulation, 87% of which were nodulated. Thus not all legumes are infected by rhizobia. Gliricidia sepium and Vigna unguiculata (cowpea) nodulate freely but nodules have never been found on roots of Cassia siamea. A Rhizobium that nodulates cowpea may not nodulate Leucaena and vice versa. Leguminous species mutually susceptible to nodulation by a particular group of bacteria constitute a cross-inoculation group. Six cross-inoculation groups were defined in the early days of Rhizobium research in addition to the cowpea group. This classification scheme is undergoing modifications based on recent research. Table 1 gives a short list of rhizobia and their hosts to illustrate the grouping of rhizobia.

Mechanisms of recognition between the microsymbiont and the host-plant have been suggested to explain specificity. (This topic is beyond the scope of this paper).

Not all symbioses fix N2 with equal effectiveness. This means that a given legume cultivar nodulated by different strains of the same species of Rhizobium would fix different amounts of nitrogen. Selection of elite strains of Rhizobium is based on this observation. Similarly, a given strain of Rhizobium will nodulate and fix different amount of N2 in symbiosis with a range of cultivars of the same plant species. Thus, different provenances of a given legume (e.g., Gliricidia sepium in ILCA's international testing) can nodulate and fix nitrogen at different levels when they are established in the same field. Also, the free-nodulating Gliricidia or promiscuous varieties of soybean can nodulate profusely and fix a great deal of nitrogen depending on the effectiveness of the rhizobial populations present.

Table 1. A short list of Rhizobium species and their corresponding hosts

Rhizobium species

Host plants

Bradyrhizobium japanicum

Glycine max (soybean)

Rhizobium fredii

Glycine max (soybean)

R. phaseoli

Phaseolus vulgaris (common bean)

R. meliloti

Medicago sativa (alfalfa)


Melilotus sp. (sweet clovers)

R. trifolii

Trifolium sp. (clovers)

R. Ieguminosarum

Pisum sativum (peas)


Vicia faba (broad bean)

"Cowpea rhizobia" group or Rhizobium sp.

Vigna unguiculata (cowpea),


Arachis hypogaea (peanut),


Vigna subterranea (Bambara groundnut)


Leucaena sp., Albizia sp.,

Azarhizobium caulinodans

Sesbania sp. Sesbania rostrata (stem nodulating)

2.4 Factors limiting biological nitrogen fixation


2.4.1 Edaphic Factors
2.4.2 Climatic Factors
2.4.3 Biotic Factors


Interactions between the microsymbiont and the plant are complicated by edaphic, climatic, and management factors. A legume-Rhizobium symbiosis might perform well in a loamy soil but not in a sandy soil, in the subhumid region but not in the Sahel, or under tillage but not in no-till plots. These factors affect either the microsymbiont, the host-plant, or both.

2.4.1 Edaphic Factors

Edaphic factors relate to the soil. The six main edaphic factors limiting biological nitrogen fixation are:

· excessive soil moisture,
· drought,
· soil acidity,
· P deficiency,
· excess mineral N, and
· deficiency of Ca, MO, CO and B.

Excessive moisture and waterlogging prevent the development of root hair and sites of nodulation, and interfere with a normal diffusion of O2 in the root system of plants. Sesbania rostrata and Aeschynomene sp. can actively fix N2 under these conditions because they are located on the plant stems, rather than on the roots.

Drought reduces the number of rhizobia in soils, and inhibits nodulation and N2 fixation. Prolonged drought will promote nodule decay. Deep-rooted legumes exploiting moisture in lower soil layers can continue fixing N2 when the soil is drying. Mycorrhizal infection has also been found to improve tolerance of plants to drought (e.g., Acacia auriculiformis inoculated with the ectomycorrhizal Baletus suillus). Mycorrhiza are symbiotic associations between fungi and plant roots. Some mycorrhizal fungi develop exclusively outside the roots; these are called ectomycorrhiza (e.g., Baletus suillus). Others, called endomycorrhiza, grow inside the roots with their vesicles and arbuscules inside the roots and with their fungal filaments extended outside (e.g., Glamus sp.). These are the vesicular-arbuscular mycorrhiza, usually referred to as VAM.

Soil acidity and related problems of Ca deficiency and aluminum and manganese toxicity adversely affect nodulation, N2 fixation and plant growth . Research work on the identification of symbioses adapted to acid soil should focus on the host plant, because effective rhizobia adapted to- soil acidity can be found naturally and can be produced through genetic manipulations.

Phosphorus deficiency is commonplace in tropical Africa and reduces nodulation, N2 fixation and plant growth. Identification of plant species adapted to low-P soils is a good strategy to overcome this soil constraint. The role of mycorrhizal fungi in increasing plant P uptake with beneficial effects on N2 fixation has been reported. Dual inoculation with effective rhizobia and mycorrhizal fungi shows synergistic effects on nodulation and N2 fixation in low P soils*. The use of local rock phosphate has been recommended, particularly in acid soils, as an inexpensive source of P. The addition of P-solubilizing microorganisms, particularly of the general Psemdamaias, Bacillus, Penicillium, and Aspergillus can solubilize rock phosphate and organically bound soil P (which constitutes 95 - 99% of the total phosphate in soils). However, the use of these microorganisms is not widespread. Some reports show nodulation response to K under field conditions. However, other investigators consider the K effect to be indirect, acting through the physiology of the plant.

* Trees are usually infected by mycorrhizal fungi in natural ecosystems in the tropics. The significance of this symbiosis in nature should be better recognised.

Mineral N inhibits the Rhizobium infection process and also inhibits N2 fixation. The former problem probably results from impairment of the recognition mechanisms by nitrates, while the latter is probably due to diversion of photosynthates toward assimilation of nitrates. Some strains of Rhizobium, and particularly stem-nodulating Azarhizobium caulinodans, fix N2 actively even when plants are growing in high-N soils (e.g., in the presence of 200 kg fertilizer N ha-1) . Application of large quantities of fertilizer N inhibits N2 fixation, but low doses (<30 kg N ha-1) of fertilizer N can stimulate early growth of legumes and increase their overall N2 fixation. The amount of this starter N must be defined in relation to available soil N.

Various microelements (Cu, Mo, Co, B) are necessary for N2 fixation. Some of these are components of nitrogenase for example Mo.

2.4.2 Climatic Factors

The two important climatic determinants affecting BNF are temperature and light.

Extreme temperatures affect N2 fixation adversely. This is easy to understand because N2 fixation is an enzymatic process. However, there are differences between symbiotic systems in their ability to tolerate high (>35°C) and low (<25°C) temperatures.

The availability of light regulates photosynthesis, upon which biological nitrogen fixation depends. This is demonstrated by diurnal variations in nitrogenase activity. A very few plants can grow and fix N2 under shade (e.g., Flemingia congesta under plantain canopy). In alley farming if hedgerows are not weeded, or if trees are planted with food crops like cassava, their nitrogen fixation and growth will be reduced due to shading. Early growth of legume trees is slow and they cannot compete successfully for light.

2.4.3 Biotic Factors

Among biotic factors, the absence of the required rhizobia species constitute the major constraint in the nitrogen fixation process. The other limiting biotic factors could be:

· excessive defoliation of host plant,
· crop competition, and
· insects and nematodes

Inoculation of Legumes

If specific and effective rhizobia are absent in a soil, or if they are present in low numbers, it is necessary to introduce the rhizobia in that soil to ensure proper nodulation and nitrogen fixation. This is called inoculation. If specific and effective rhizobia are present in a sufficient number, there will be no need to inoculate the legume. In agrisystems, whenever one is not sure of the presence and effectiveness of the native rhizobia, it could be necessary to inoculate the legume with an adequate strain of rhizobia.

How one can determine the need for inoculation? There are some simple tests: Are nodules absent or sparse on an uninoculated young plant growing in a low-N soil? (This is normally accompanied by plant N deficiencies). Or, are nodule sections white or green? (This is an indication of poor effectiveness).

A more accurate relative effectiveness trial will provide more precise information. The trial, in a simple term, consists of growing the legume with and without fertilizer N while controlling all other limiting factors. The relative effectiveness ratio (RE) is then calculated. RE is defined as: dry weight of unfertilized plants × 100/dry weight of fertilized plants. If the value of RE is more than 5, the inoculation is not required.

When the rhizobia in a soil are infective (i.e., capable of colonizing and nodulating a legume) but poorly effective, they constitute a barrier to the successful exploitation of Rhizobium inoculants. Introduced rhizobia must therefore be more aggressive and competitive as nodulators than the native strains. Inoculant rhizobia usually persist in the soil for long periods, particularly when the host is cultivated frequently or is permanent. Persistence of a strain is desirable because it obviates the need for inoculation in subsequent years, assuming inoculant strains maintain their original effectiveness.

Inoculation with rhizobia is usually recommended for newly introduced legumes. Most positive responses to inoculation are confined to crops which have specific requirements for Rhizobium, (e.g., Leucaena leucocephala, American varieties of soybean). Indigenous legumes seldom respond to inoculation with introduced rhizobia because they nodulate with resident strains, even if these native rhizobia are not the most effective ones.

Inoculation with rhizobia should be considered as an exceptional farming practice rather than the rule. In Australia and the USA, inoculation has played a vital role in legume production. But in developing countries, the practice is not widespread. The major drawback to inoculation technology is the wide variability in yield responses in time and space for a given Rhizobium-legume symbiosis. Responses can vary from no response, and sometimes negative responses, to positive yield increases. Response to inoculation with a strain of Rhizobium vary with sites, legume cultivars, and the form of inoculant. Changes in climate, such as Africa's long droughts in recent years, and management factors including cropping systems and inoculant handling will also introduce variability in response to inoculation. Local rhizobia are not necessarily better inoculants than exotic strains.

All these considerations call for a substantial research support system capable of defining the most appropriate inoculants and procedures for each site and probably for each cropping season as well. The use of freely nodulating legumes will be much easier in this respect.

Inoculation procedures are detailed in Volume 1 of this training manual (see Appendices).

Defoliation, Crop Competition, and Pests

Defoliation (e.g., pruning and lopping) decreases the photosynthetic ability of legumes. It impairs N2 fixation and can lead to nodule decay. For perennial legumes, nodule decay sheds a high number of rhizobia in the root zone. When new roots develop in subsequent vegetative cycles, nodulation of the legume is expected to improve. Scientists at IITA have observed that uninoculated Leucaena leucocephala nodulated very sparsely the first year and showed nitrogen deficiency symptoms. After a number of years nodulation improved and N deficiency symptoms disappeared.

Intercropping legumes with non-leguminous crops can result in competition for water and nutrients. This competition can affect N2 fixation negatively. However, it has been shown that when mineral N is depleted in the root zone of the legume component by the non-leguminous intercrops, N2 fixation of legumes may be promoted.

Insects and nematodes have also been reported to interfere with nodule formation, development, and functions.

2.5 Estimation of nitrogen fixation


2.5.1 Short-term Estimation of BNF: Acetylene Reduction Assay
2.5.2 Medium-term Estimation of BNF: N-solute Analysis of Xylem Exudate


From the biochemical reactions of BNF presented in section 2.1, it is evident that N2 fixing systems contribute to the quality and quantity of agricultural production. Measurement of BNF can provide information on whether actual N2 fixation in adequate. We discuss below two simple methods of BNF estimation. Measurement of BNF is a more reliable method than nodule counting, nodule weighing, or assessment of leghemoglobin.

2.5.1 Short-term Estimation of BNF: Acetylene Reduction Assay

Nitrogenase not only catalyzes the reduction of atmospheric N2 to NH3, but can also reduce acetylene (C2H4). The acetylene reduction assay (ARA) is carried out on detached nodules, detopped roots, or whole plants in a closed vessel containing 10% acetylene. A gas chromatograph is used to determine the amount of ethylene formed. Data are usually expressed as nanomoles or micromoles of ethylene produced per hour per plant or per weight unit of nodules. The acetylene reduction assay provides an instant measure of nitrogenase activity (but not necessarily of N2 fixed) under the experimental conditions.

For long-term estimates, a series of measurements must be performed to include diurnal, daily, and seasonal changes. Variation in light intensity, temperature, and moisture in the field will increase the level of variation of nitrogenase activity and will reduce the significance of integration of short-term assays. A problem that is inherent in ARA is the need to calibrate the rates of ethylene production with the actual rates of N2 fixation. The commonly used ratio of 3:1 for acetylene reduced per N2 fixed is not always valid. Also, nitrogenase activity of some legumes declines considerably once nodules or roots are detached from the rest of the plant. For plants with long roots, it is difficult to collect all the nodules. To minimize this limitation, the plants are confined to open ended chambers and ARA is done in situ.

2.5.2 Medium-term Estimation of BNF: N-solute Analysis of Xylem Exudate

N-solute analysis of xylem exudate is a medium-term type of estimate because it involves the integration of more than one hour of events. The underlying principle is based on the fact that nitrogen from BNF can be transported to the leaves in the form of (1) ureides, allantoin and allantoic acid, or (2) asparagine and glutamine. In agricultural soils, where nitrate is the most readily available form of N for plant growth, the solutes derived from soil mineral N will contain principally free nitrate and organic products of nitrate reduction in the roots. Correlations can be established between the N2 fixed nitrogen in forms (1), (2), and soil-derived N. Using these correlations, it should be possible to assess N2 fixation, or at least to obtain an index of BNF by collecting and analyzing plant sap for the above-mentioned N compounds.

The methods are simple and have been used successfully in ureide legumes. Solute analysis can be used in farmers' fields because it is virtually non-destructive. It is also relatively inexpensive. Repeated measurements are also required to fully integrate measurements of total N fixed over a long period of time. Table 2 presents the occurrence of ureides in xylem sap of nodulated legumes.

Table 2. Occurrence of ureides in xylem sap of nodulated legumes (Peoples et al. 1989)

Species in which ureides are major components of solute N (a)

Species in which ureides have been defected as a minor component (b)

Species in which ureides have not been detected

Albizia Iophantha

Albizia falcataria

Acacia alata

Cajanus cajan

Bossaiae aquifolium


auriculiformia

Calopoganium caeruleum

Erythrina variegate


extensa

Centrosema spp.

Flemingia congesta


insauvis

Codariosalyx gyroides

Gliricidia sepium


pulchella

Cyamopsis tetragonoloba

Pisum arvense

Arachis hypogaea

Desmodium discolor

Sesbania rostrata

Baubinia spp.


renssonii


sesban

Caesalpinia


uncinatum


calothyrsus


Calliandra spp.


Stylosanthes bamata

Vicia ervilia

Cicer arietinum

Glycine max


sativa

Clitoria spp.

Hardenbergia spp.

Viminaria juncea

Derris elliptica

Lablab purpureus


Juncea spp.

Macroptilium atropurpureum


Lathyrus cicera

Macrotyloma uniforum



sativus

Pueraria javanica


Leucaena spp.

Phaseoloides


Lens culinaris

Phaseolus vulgaris


Lotus corniculatus


eunatus


Lupinus albus

Phophocarpus tetragonolubus



angustifolius

Tedbegi spp.



cosentinii

Vigna angularis



mutabilis


mungo


Medicago minima


radiata



sativa


triloba


Mimasa pigra


unguiculata


Pisum sativum


umbellata


Sesbania grandiflora

Voandzeia subterranea


Trifolium paratense




subterraneum




repens



Vicia monantha




faba



Zornia spp.

(a) 40% or more of total N of xylem sap estimated] to be in ureides..
(b) 10-25% of total N of xylem sap collected from glasshouse-grown or field plants estimated to be in ureides.

2.6 How to increase BNF and N2 fixing ability

Biological N2 fixed represents N gain and determines inorganic N fertilizer savings in cropping systems. Legumes can fix more than 250 kg N ha--1. However, the amounts of N2 fixed can vary considerably in time and space The nitrogen fixation process is influenced by factors such as:

· presence and effectiveness of rhizobia, pest damage,
· plant genotype and age,
· plant and rhizobia interactions,
· changes in soil physiochemical conditions, and
· various management practices such as tree pruning or pesticide application that can affect both symbiotic partners.

Four common approaches to enhance biological nitrogen fixation are:

· inoculation with proven strains (covered above),
· microbial screening for improved strains,
· host-plant screening and breeding, and
· adoption of cropping systems and cultural practices.

Microbial Screening

There are collections of effective rhizobia located at centers around in the world for most, if not all, legumes used in agriculture (Takishima et al, 1989). These strains may be screened to identify the most effective and competitive one(s) for a given agroecosystem Once elite strains have been identified, the legume under consideration is inoculated. Instructions on inoculant use are usually given by the manufacturers. Seed inoculation using peat inoculant is the most commonly used method. However, studies are under way to assess the effectiveness of post planting inoculation as a corrective measure Dual inoculation of rhizobia and mycorrhizal fungi has proven beneficial in some cases.

Host-plant Screening and Breeding

A screening of legume plants, with high N2-fixing components can be carried out Breeders have developed plant varieties with promiscuous nodulation to obviate the need for inoculation with rhizobia In some laboratories in the USA, plants that do not nodulate with indigenous rhizobia but only with introduced "super" strains are being developed

There are still many unexploited legume-Rhizobium symbioses in the world. The potential benefit of screening these symbioses is underscored by the fact that only about 0.5% of existing leguminous species are presently used for agricultural purposes.

Cropping Systems and Cultural Practices

It is evident that inclusion of N2-fixing components in cropping systems will increase N inputs in agrisystems. Cultural practices can control some of the above-mentioned factors which limit BNF. Mulching, for instance, can control weeds and fluctuations of soil moisture and temperature. Liming can eliminate soil acidity, and Al and Mn toxicities.

2.7 Summary

Since nitrogen is commonly the most limiting plant nutrient in arable farming in the tropics and also the most expensive element as a mineral fertilizer, biological nitrogen fixation (BNF) holds great promise for smallholder farmers in sub-Saharan Africa. Alley farming systems which use leguminous woody species in the hedgerows can reduce or eliminate farmers' needs for commercial N fertilizer.

Biological nitrogen fixation is the process of capturing atmospheric nitrogen by biological processes. It is accomplished by certain microorganisms and plant-microbe interactions. Legumes are N-fixing systems that have long been used for biological nitrogen fixation in agriculture.

Biologically fixed nitrogen can be estimated using the acetylene reduction assay method, xylem exudate analysis, or by other methods.

A number of edaphic, climatic, and biotic factors inhibit N2 fixation Among these, the absence of specific and effective rhizobia in the soil is the most important. The amount of biologically fixed nitrogen can be enhanced by different methods, including inoculation with proven strains, screening for improved microbial and host-plant materials, and introduction of improved cultural practices.

2.8 Feedback exercises (Find out answers from the text)

1) Provide a brief answer to each of the following:

a. What is nitrogenase ?
___________________________

b. Name 4 microorganisms in which nitrogenase may be found.
________________________
________________________
________________________
________________________

c. What is the role of ATP in biological nitrogen fixation?
________________________

d. What specific functions do flavodoxin or ferredoxin perform in biological nitrogen fixation?
________________________

2) a. Complete the missing components of the mechanism of N2 fixation as shown below:

N2-------> ...?... --------> amino acids -------> ...?...
+ ATP
+ ...?...
+ ...?...

b. Match the names of the Rhizobium species (on the left) with the appropriate host-plants (on the right).

. Cowpea rhizobia group

Medicago sativa (Alfalfa)

. Rhizobium fredii

Leucaena sp.

. Rhizobium meliloti

Glycine max (Soybean)

3) Circle T for true and F for false.

a. Excessive moisture in the soil inhibits biological nitrogen fixation primarily by creating iron toxicity.

T

F

b. Under dry conditions, deep-rooted legumes behave exactly the same way as shallow-rooted legumes in terms of the amount of nitrogen fixed

T

F

c. Phosphorus deficiency reduces plant growth and nodulation, thereby adversely affecting nitrogen fixation.

T

F

d Excess mineral nitrogen in the soil will enhance nitrogen fixation by legumes because it increases plant vigor.

T

F

e. Because biological nitrogen fixation normally occurs in the roots, light availability will have no impact on N2 fixation.

T

F

f. Different symbiotic systems have different tolerances to temperatures.

T

F

g. Inoculation is the process of introducing specific and effective rhizobia in the soil to ensure nodulation and nitrogen fixation.

T

F

h. Defoliation of pruning increases nitrogen fixation by creating a greater demand for nitrogen by the plants.

T

F

4) What are the two major benefits of growing leguminous species in the hedgerows of an alley farm, as compared with non-leguminous hedgerows? (select 2 from the list)

a. Leguminous hedgerows protect the food crops from wild animals.

b. Prunings from leguminous hedgerows are source of nutritious protein-rich feed for livestock.

c. Leguminous hedgerows prunings have insecticidal value and their incorporation in soils protects plants from soil-borne pests.

d. Prunings from leguminous hedgerows provide cheap nitrogen for food crops.

e. Leguminous hedgerows create a cool microclimate which indirectly benefits the associated crops.

2.9 Suggested reading

Adetiloye, P.O. and Adekunle, A.A. 1989. Concept of monetary equivalent ratio and its usefulness in the evaluation of intercropping advantages. Tropical Agriculture (Trinidad), 66, 377-41.

Dommergues, Y. et Mangenot, F. 1970. Ecologie microbienne du sol. Masson & Cie, Ed. 120 Boulevard Saint Germain, Paris VI.

Elkan, Gerald H. 1987. Symbiotic Nitrogen Fixation Technology. Marcel Dekker, Inc. N.Y. 10016, USA.

Ngambeki, D.S. 1985. Economic evaluation of alley cropping Leucaena with maize-maize and maize-cowpea in southern Nigeria. Agricultural Systems, 17, 243-58.

Raintree, J.B. and Turay, F. 1980. Linear programming model of an experimental leucaena-rice alley cropping system. IITA Research Briefs, 1, 5-7.

Sumberg, J.E., McIntire, J., Okali, C and Atta-Krah, A. 1987. Economic analysis of alley farming with small ruminants. ILCA Bulletin, 28, 2-6.

Verinumbe, I., Knipscheer, H. and Enabor, E.E. 1984. The economic potential of leguminous tree crops in zero-tillage cropping in Nigeria: A linear programming model. Agroforestry Systems 2, 129-38.

2.10 References

Peoples, M.B., A.W. Faizah, b. Rerkasem, and D.F. Herridge 1989. Methods for Evaluating Nitrogen Fixation by Nodulated Legumes in the Field. ACIAR Monograph No. 11, vii + 76 p., Canberra.

Takishima, Y., Shimura, J. Ugawa, Y., and Sugawara, H. 1989. Guide to World Data Center on Microorganisms with a List of Culture Collections in the World. 1st edition. Saitama, Japan: WFCC World Data Center on Microorganisms.


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