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  Section: General Biotechnology / Plant Biotechnology
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Biological Nitrogen Fixation

Symbiotic N2 Fixation
There are some microorganisms which establish symbiotic relationships with different parts of plants and may develop (or may not) special structures as the site of nitrogen fixation. Microsymbionts are given in Table 11.1. Non-nodule forming diazotrophs, for example, Azotobacter, Beijerinckia, Derxia are known to be intimately associated with the roots of certain plants. Azotobacter paspali is restricted to the roots of the tropical grasses, Paspalum notatum and rare to other species of Paspalum or other genera (Dobereiner, 1970). Beijerinckia shows host specificity with sugarcane root. On the other hand, Azospirillum is known to be associated with the roots of corn, wheat, sorghum, Digitaria decumbens, Panicum maximum and Melinis multiflora, and a large number of mono-and dicot plants (Elmerich, 1984).

Azospirillum is Gram-negative aerobes ; it is curved and rod shaped, and has polar flagellum. Bacteria are associated with the grass roots in such a way that a gentle washing do not dislodge the nitrogen metabolizing activity (Alexander, 1977). Based on acetylene reduction it has been calculated that A paspali contributes 15-93 kg N/ha/annum on P. notatum roots, and Beijerinckia assimilates about 50 kg N/ha/annum on sugarcane root (Dobereiner et al, 1973). Other bacteria on corn roots may fix about 2.4 kg. N/h/day (Bulow and Dobereiner, 1975). Azospirillum increases yield of cereals amounting to a saving of fertilizer nitrogen equivalent to 20-40 Kg/ha.



Non-Symbiotic N2 fixation




Ecology of diazotrophs


Special features of diazotrophs



Sites of N2 fixation



Nitrogenase and reductants



Presence of hydrogenase



Self regulatory systems


Mechanism of N2 fixation

Symbiotic N2 fixation


Establishment of symbiosis



Host specificity and root hair curling



Infection of root hairs



Nodule development



Nodule development and maintenance


Factors affecting nodule development


Mechanism of N2 fixation in root nodules

Genetics of diazotrophs


Nod genes


Nif genes



Nif gene cloning


Hup genes

In addition to these bacteria, Frakia, Rhizobium sp. and cyanobacteria undergo symbiosis by getting established inside the plant tissues and may or may not develop special symbiotic structures.

The classical examples of symbiotic association developed by Rhizobium sp. are found in about 13,000 or more leguminous plants, both cultivated and non-cultivated herbs, shrub and trees. Among leguminosae, the largest number of plants is in papilionoidae. Moreover, species of Anabaena, Nostoc, Tolypothrix, etc. develop symbiotic association with fungi (symbiotic structure is lichen), bryophytes, pteridophytes, gymnosperms and angiosperms and fulfill the requirement of nitrogen deficiency (Table 11.1). However, actinorrhizic nodules are developed by Frankia, a member of Actinomycetes, on roots of about 170 species of woody dicot non-leguminous plants from 15 genera belonging to temperate trees like Alnus, Myrica, Casuarina, etc. (Goodfellow and Williams, 1983). Nodules; are of two types: (i) Alnus type where nodules show dichotomous branching to form a corraloid root, and (ii) Myrica/Casurina type in which case the apex of each nodule produces a normal but negative geotropic root. The function of nodules is to facilitate gas diffusion to the nitrogen fixing endophyte in the nodule under low O2 tension (Becking, 1982).

Table 11.1. Symbiotic microorganisms.



Symbiotic structures






Azotobacter paspali

No special structure develops, intimately associated with roots

Paspalum notatum, roots of corn,wheat, sorghum, Digitaria decombense

Azosipiillum amazonense

A. brasilense

A. ligoferum

Beijerinckia sp.

Derxia sp.

Rhizobium sp.

Root nodules

Leguminous plants, and non-leguminous plant (e.g. Trema canabaena)


Actinomycetes Frankia sp

Root nodules

Non-leguminous angiosperms eg. Alnus, Casuarina, Myrica, Discaria





Anabaena, Nostoc and Tolypothrix

Lichens (Collema, Peltigera, Usnia)


Ascomycetous and basidiomycetous fungi

Nostoc sp.

Bryophytes: Anthoceros, Blasia

Anabaena azollae

Pteridophytes : Azolla

A. cycadae

Coralloid roots

Gymnosperms : Cycas


Angiosperm : Gunnera

*Besides Rhizobium sp., all the bacteria listed are associate symbionts; they are heterotrophs in their habit.

Rhizobium is a free-living, Gram negative nonsporulating, aerobic and motile rod shaped bacterium (0.5-0.9mm x 1.2 - 3mm) which resides in soil and is capable of infection, nodulation, establishing symbiosis and N2 fixation. It grows on organic nutrients. Rhizobia are more prominent in the rhizosphere of leguminous plants. Based on cross-inoculation studies, six species of Rhizobium are defined according to the legume host(s) which they nodulate (Table 11.2). A cross-inoculation group refers to a collection of leguminous species that develop nodules on any member of that particular plant group. Therefore, a single cross inoculation group ideally includes almost all host species which are infected by an individual bacterial strain. Six inoculation groups have been specified so far.

Elkan (1985) grouped the nodule forming bacteria into the following two:

Genus I:

Rhizobium - it consists of the fast growing and the flagellated strains. The species are :
R. leguminosarum biovars. trifoli : host - Trifolium, and R. leguminosarum biovars . phaseoli : host - Phaseolus
(ii) R. meliloti :
host - Lotus.

Genus II.

Bradyrhizobium - it contains the slow growing or sub-polar flagellated strains.
B. japonicum : host - Glycine
(ii) Brady rhizobium sp.
: host - Cicer, Vigna, Cajanus.

Establishment of Symbiosis
Establishment of Rhizobium inside the host root and development of nodules are a complex process which follow many events such as recognition and infection of host root, differentiation of nodules, proliferation of bacteria and conversion into bacteroids in nodules. These steps are briefly described as below :

Host specificity and curling of root hairs

A variety of microorganisms reside in close vicinity, the rhizophere, of roots. Depending on environmental conditions and host susceptibility, the phenomenon of host recognition by the specialized microbe is achieved. The level of specialization for symbiosis differs in different microbial groups and even in the same group as well. As a result of host recognition, Azospirillum sp. in some cases are intimately associated with their host and have been isolated from the rhizoplane also. The apparently differentiated structures are not formed, but pictures of root hair deformation are known. Moreover, Azospirillum can also invades cortical and vascular tissue of host, and enhances the number of lateral root hairs. This results in an increase in mineral uptake, which may be attributed to phytochrome production rather than N? fixation (Lin et al, 1983; Okon, 1985). Host specificity in Azospirillum has also been noted but it differs from that of Rhizobium.

Table 11.2. Species of Rhizobium and cross inoculation groups of host plants.

Rhizobium sp.

Host genera

Cross inoculation

R. japonicum


Soybean groups

R. leguminosarum

Pisum, Lathyrus, Vicia, Lens

Pea group

R. lupini

Lupinus, Ornithopus

Lupin group

R. meliloti

Melilotus, Medicago, Trigonella

Alfalfa group

R. phaseolus


Bean group

R. trifolii


Clover group

Other species of Rhizobium

A rack is, Crotalaria, Vigna, Pueraria

Cowpea group

Host plant secretes exudates in rhizosphere ; subsequently compatible strains of rhizobia are stimulated over the other microbes in soil. Root exudates contain growth stimulating substances like biotin, thiamine, amino acids, etc. Bacteria grow near the root surrounded by mucigel. Mucigel denotes microbial cells and their products together with associated microbial cells and mucilages, organic and inorganic matter in the rhizosphere of root region. The initial host response is observed as root curling which is known as Shepherd's Cook (Fig. 11.6.A). Root curling takes place due to secretion by Rhizobium of a curling factor which includes cytokinin, polymixin B, etc. (Dart, 1974). 

According to current theories the host-symbiont specificity is governed by a specific plant protein called lectins (phytoagglutinins) involved in recognition of compatible symbiont. It is supplemented by secretion of specific poly-saccharides by the symbiont termed as callose which helps in attachment of rhizobia on host root surface (Bahlool and Schmidt, 1974; Dazzo and Hubbell, 1975).

Infection of root hairs
Rhizobial aggregates have been observed at distinct sites on curled root hairs (Fig. 11.6 A II). Nutman (1956) has suggested that the infection thread is formed by a process of invagination of the hair cell walls in the region of curiing. This process of invagination is being repeated at each cell penetrated by infection thread. Root hair wall invaginates until it develops a tube like structure.
  Root nodule formation in a leguminous plant. A-transverse section of a root showing root hairs and its invasion by infection threads of Rhizobium sp.; B-vertical section through nodules; C-a cell of the infected host filled with Hiizobia; D-various shape of bacteroids; E-root system of a leguminous plant showing numerous nodules.

Fig. 11.6. Root nodule formation in a leguminous plant. A-transverse section of a root showing root hairs and its invasion by infection threads of Rhizobium sp.; B-vertical section through nodules; C-a cell of the infected host filled with Hiizobia; D-various shape of bacteroids; E-root system of a leguminous plant showing numerous nodules.

Deformed hairs are penetrated in the first stage of infection (Fig. 11.6 A III). Not all, only a few proportion (about 5%) of young root hairs develop nodules. Following the penetration, a hypha like infection thread is formed. The infection threads resemble to invading fungal hyphae. It is a unique structure which contains a cellulose sheath deposited by the host cell enclosing a strand of hemicelluiosic substance in which the bacteria are embeded (Schaede, 1940). If the population of rhizobia is even in the infected tubes, it can be observed under light microscope also. The infection thread grows towards root cortex and ramify throughout the central part of cortex (Fig. 11.6 A IV). It is noteworthy that plasmoptysed hairs have not been observed to form infection threads, although they can be invaded by bacteria. Plasmoptysis may be an important mechanism of root exudation (Nutman, 1965).

Nodule formation
As the infection thread continues to grow through the root tissue, inner cortical cells are stimulated by bacteria through growth hormone to divide and form an organized mass of infected plant tissue which protrudes from the root surface as a visible nodule (Fig. 11.6B). Rhizobia are released from the infected threads, within some of the cortical cells of nodule and multiply thereby rapid cell division, and ultimately occupy the major central portion of root nodule. The peculiar feature of cells of central nodule is that it contains tetraploid chromosome number. The chromosome doubling of nodule cells may be attributed to secretion of stimulating chemicals by rhizobia.

In the nodules the rhizobia are found to be enclosed in a membrane derived from hypertrophied cells of the host (Fig. 11.6C), where they multiply into 4 -6 in number, and finally become enlarged and pleomorphic to form bacteroids (Fig. 11.6D). In morphology, bacteroids appear as swollen, irregular, star shaped, club-shaped, branched or Y-shaped structures. Size and shape of the bacterioids vary in different species of Rhizobium. On accomplishment of nodule formation process, the root hairs of plant contain a clusture of nodules (Fig. 11.6E). 

Nodule development and maintenance
Many plant and bacterial genes express in an sequential manner during nodule development and maintenance in legumes. Legochi and Verma (1980 studied the Rhizobium - legume symbiosis. They found the expression of plant genes and production of nodule specific protein during certain stages of nodules. They termed these proteins as 'nodulins'. On the basis of functions, nodulins have been categorized into three (i) nodule structure maintaining proteins, (ii) bacteroid function (N2 - fixation) supporting proteins and (iii) proteins (enzymes) expressed in specific nitrogen assimilation and carbon metabolism (Fuller et al, 1983). Moreover, on the basis of structural resemblance nodulins are divided into two : C-nodulins and S- nodulins. C-nodulins (common nodulins) are the proteins common to all nodules, while S-nodulins are referred as species specific nodulins and not found commonly in all species.


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