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3.3 Nitrogen-fixing associations

What is the range of associations between nitrogen-fixing microorganisms and plants?

Nitrogen gas (N2) constitutes nearly 80% of the atmosphere, but in this form nitrogen is not available to plants. Several groups of soil bacteria convert nitrogen gas into ammonium (NH4+), which is a form of nitrogen that can be used directly by plants. This transformation is catalysed by the bacterial enzyme nitrogenase. Very few bacteria have the capacity to activate the nitrogenase enzyme system, and those that do occur as free living bacteria in soil, as loose associations with root surfaces or within highly specialised, symbiotic associations with plants.

Bacteria in the genus Azospirillum live in a loose association with root surfaces of grasses and other plants. This contrasts with the highly specific associations that form between cyanobacteria and fungi in lichens. Highly specific nitrogen-fixing associations are also formed between some tree species and bacteria (actinomycetes) in the genus Frankia. The more primitive gymnosperms such as cycads also form characteristic associations with cyanobacteria. The most widely studied of all nitrogen fixing associations are formed between legumes and bacteria from the family Rhizobiaceae.

The biological transformation of N2 gas to ammonium is equivalent to the industrial transformation of N2 that requires excessive application of heat and pressure. This highlights the uniqueness of biological nitrogen fixation and its practical benefit.

What is ‘associative’ nitrogen fixation?

Nitrogen-fixing bacteria that live in loose associations with roots are called ‘associative’ nitrogen fixers. Associative nitrogen fixing bacteria grow on the surface of roots and may also colonise the outer layers of a root by entering between epidermal cells.

A common genus of associative nitrogen fixing bacteria is Azospirillum. Species of Azospirillum are not all equally effective at converting atmospheric nitrogen to ammonium. There is conflicting evidence about the extent to which nitrogen fixed by these bacteria is available to plants. Even for the species of Azospirillum that are very effective at fixing atmospheric nitrogen, the nitrogen fixed is not immediately available to the plant. It is not until the bacteria die and are mineralised that the nitrogen becomes available to the plants.

Nitrogen fixed by Azospirillum is not immediately available to plants because of the complex processes of ammonium transfer through the bacterial cell wall. Ammonium excess to the requirements of the bacterium can be transported to the outside surface of the cell wall. However, another process can transfer the ammonium back into the cell, slowing the transfer of ammonium to the plant. Some mutant strains of Azospirillum are effective at permanently transferring ammonium from the bacteria (Christiansen-Weniger and Van Veen 1991).

The movement of molecules into and out of bacteria and fungi are complex processes. In the case of Azospirillum, an understanding of these processes shows why nitrogen is not immediately available to plants from bacteria that fix nitrogen in the root zone, at least while the bacteria are alive.

The benefits of associative nitrogen fixers have been estimated in agricultural soils. The quantity of nitrogen fixed varies greatly. In very nitrogen deficient soils, the quantities of nitrogen fixed are not usually sufficient to overcome nitrogen deficiency in agricultural plants.

How do associations between legumes and nitrogen fixing bacteria function?

Legumes are important plants in both natural as well as managed ecosystems. The majority of legumes form symbiotic associations with nitrogen-fixing bacteria. As a consequence of these associations, distinctive structures (called nodules) form on the roots of legumes. The bacteria multiply within the nodule cells.

Many kilograms of nitrogen per hectare can be converted every year by agriculture plants including crop (e.g. soybean) and pasture (e.g. clover) plants because of their associations with nitrogen-fixing bacteria. These associations are of great importance as a substitute for nitrogen fertiliser. Nitrogen fixing symbioses are also common in natural ecosystems.

Bacteria that form associations with legumes live in the soil as part of the microbial community. However, they do not fix nitrogen directly in the soil. Nitrogen fixation only occurs when bacteria form a nodule on a legume root. When an appropriate legume is present, only one in a million bacteria are necessary to form a nodule. In forming a nodule, the bacterium undergoes a highly specific interaction with the plant, involving several stages and complex signals between the bacterium and plant (Sprent 1989).

Associations between particular bacteria and their legume host plants are usually highly specific (Table 49). For example, the bacteria that fix nitrogen in association with species of clover cannot fix nitrogen in association with species of acacia. Nevertheless, the same bacteria can form associations with different clover species and occasionally different bacteria will infect the same plant. However, nodules generally contain only one strain of bacterium that multiplies to fill the central core of nodule cells.

Although there is considerable specificity among root nodule bacteria and legumes, it is interesting that the same bacteria form nitrogen-fixing associations with lupin (Lupinus spp.), a crop legume and serradella (Ornithopus spp.), a pasture legume. Because the architecture of both the roots and shoots of these two plants is very different, the bacteria which colonises both are evidently highly adaptable.

The shape and size of nodules varies considerably, with each determined by the plant, not the bacteria. In some plants, nodules can continue to grow throughout the life of the plant, as for example with clover. Where this occurs on perennial species, layers of new nodule tissue are added annually. In contrast, nodules formed on other legumes have a limit to their growth and are termed determinant nodules. Soybean is an example of a legume that forms determinate nodules.

The bacteria that form symbiotic associations with legumes differ in their ability to fix nitrogen (Sanginga et al. 1989). The effectiveness of these bacteria can also be influenced by soil conditions. Therefore, when planting agricultural legumes it is necessary to select the most effective bacteria that will do best in the environment. This is also true when planting tree species for revegetation and forestry projects.

The bacteria that fix the most nitrogen when in association with a legume are not necessarily those that form the most nodules. Conversely, it is not uncommon to find nodules formed on agricultural plants by bacteria that are ineffective at fixing nitrogen. In soils where there are different strains of bacteria that can nodulate the same legume, many factors influence the success or failure of nodulation. Therefore, the full potential for nitrogen fixation is not always achieved, even if highly effective strains of bacteria occur in the soil.

Diversity among root nodule bacteria

There is considerable diversity among root nodule bacteria (McInnes et al. 2004). Bacteria that form nodules on legumes differ in the rate at which they multiply on artificial laboratory media. Acacias form nodules with groups of bacteria with very different characteristics and this can affect the detection of bacteria on growth media if the rapidly growing bacteria overgrow those that grow more slowly.

There is a difference in the DNA structure among root nodule bacteria. In some, but not all, DNA is present as circular segments, separate from the main chromosome. These segments, called plasmids, contain the genetic information required to initiate symbiotic nitrogen fixation. Plasmids can replicate separately from the chromosome and transfer from one bacterial cell to another.

Genetic basis for nodulation and nitrogen fixation

The location of genes on the DNA that code for different stages in the nodulation process illustrates complex interactions that take place between legumes and root nodule bacteria during nodulation. Separate regions of bacterial DNA are responsible for recognition of the legume root surface, initiation of root hair curling, initiation of the infection thread formation, and root cell multiplication to form the nodule.

Bacteria present in the rhizosphere stimulate the plant to produce molecules called flavanoids which are released in root exudates. In turn, these molecules can, to varying degrees activate the nodulation genes in the bacteria. For successful nodulation to occur, the flavanoid molecules need to be present in sufficient quantities to induce a response in the bacteria. Some plants are better able to do this for some bacteria than others (Howieson et al. 1992). In this example, the maximum activity of flavanoids produced by Medicago murex occurred at pH 6.0 whereas flavanoids produced by Medicago littoralis were most active at a higher pH. This response partly explains why nodulation changes with soil pH for some legumes. Even though the bacteria may grow well at a certain soil pH, the lack of production of exudates that will initiate nodule formation may hinder their ability to form a nodule (See Halverson and Stacey 1984).

Legumes in natural ecosystems grow in soil containing indigenous, diverse communities of root nodule bacteria. Similarly, highly managed agricultural systems may contain many different strains of bacteria that have been introduced with agricultural legumes. High diversities of root nodule bacteria can result from a lengthy history of growing different legume species on the land, multiple introductions of commercial inoculant root nodule bacteria, and ongoing genetic diversification of bacteria in soil.

An analysis of the range of strains of root nodule bacteria that occur in Oregon pastures illustrates the level of genetic diversity that is possible (Bottomley and Dughri 1989). In this study, the bacteria were characterised using serological methods with four distinct serogroups identified and one most abundant in the surface 10 cm of the soil profile. Subsequent investigations using DNA analysis confirm the diversity of strains of rhizobia in soils.

When bacteria selected for a particular legume are introduced into a soil, they have to survive as part of the community of soil bacteria. Otherwise, the only way they will be maintained in the soil is by regular re-introductions. The newly introduced bacteria also need to compete with existing strains of similar bacteria for nodulation sites on the legume root. For example, the strain of bacterium that was one of the original inoculants for soybean in the USA was subsequently found to be less effective at fixing nitrogen than some strains introduced later. The original bacterium was very competitive at forming nodules on soybean in comparison to the more recently identified effective strains. It is difficult for a newer bacterium to outcompete older bacteria that have already colonised the soil and are highly competitive at forming nodules (see Denton et al. 2002). This is clearly a major problem for the introduction of strains that are more effective at fixing nitrogen. A similar obstacle occurs when two clover species are grown in the same pasture. Nitrogen fixation occurs most effectively for each with different strains of Rhizobium leguminosarum biovar trifolii. However, it is not possible to ensure that each clover species is nodulated by the most appropriate strain of bacterium. The result could be that neither clover species fixes nitrogen according to its potential.

Bacteria that form legume symbioses can survive in a soil in the absence of suitable plants. Once the roots of a suitable plant reappear, the bacteria can re-initiate the symbiotic association. Some bacteria can survive without a plant for longer than others can. Even if nodule formation does not occur as soon as the plant reappears, the plant can stimulate the growth of the bacteria and make it more likely that nodulation takes place when other soil conditions are suitable.

Which non-legume plant species form nitrogen-fixing associations?

Frankia is a genus of bacteria (actinomycetes) that has the capacity to fix nitrogen gas in association with several tree species. Examples of such trees include the genera Casuarina and Allocasuarina in the family Casuarinaceae and Alnus in the family Betulaceae.

The associations formed by species of Frankia are generally host-plant specific, but the same bacteria can nodulate closely related plant species (Reddell and Bowen 1986). Frankia associations with plants range from those that are highly effective to those that are relatively ineffective at fixing nitrogen (Nagashima et al. 2008).

Nitrogen deficiency in Casuarina can be remedied by inoculation with strains of Frankia that are effective at fixing nitrogen (Reddell and Bowen 1985). Two of the five sources of Frankia studied were effective at increasing growth of C. equisitifolia. Another was highly effective with C. cunninghamiana.

The association between Frankia and roots of their host plant is highly specific. The infection process is initiated through molecular signalling between the bacteria and root as for legume symbioses. When there is a compatible combination of bacteria and plant, molecular communication leads to the formation of a nodule on the plant root that develops a colony of bacteria within it. The bacteria stimulate the plant to form nodule tissue. Nodules can live for a long time, with a new layer of actively growing root cells and bacteria added each year. The bacteria fix nitrogen gas that diffuses through the walls of the nodule cells; the fixed nitrogen is transferred into the root cells and from there into the rest of the plant through the vascular system.

A variety of other associations also occur between nitrogen-fixing organisms and plants. For example, Macrozamia riedlei, a member of the family Cycadaceae, forms an association with nitrogen fixing cyanobacteria. This plant forms highly branched roots that contain the cyanobacteria.

Summary Points (3.3)

• Non-symbiotic nitrogen fixation occurs both in soil and in close association with roots.

• Symbiotic nitrogen fixation occurs in close association with roots.

• Various degrees of specificity occur between plants and root nodule bacteria in both legume and non-legume nitrogen fixing symbioses.

• Root nodule bacteria differ in their effectiveness in fixing nitrogen.

• The processes involved in the formation of nodules on legumes and non-legumes are generally highly specific and are finely co-ordinated by molecular signalling between the bacteria and the plant.

• The bacteria that form symbioses with plants are diverse and can modify in their important symbiotic characteristics as a result of exchange of genetic information among soil bacteria or loss of this information.

• Symbiotic nitrogen fixation occurs in natural ecosystems where appropriate plant species are present and in managed agricultural systems where legume crop and pasture species are grown without the addition of nitrogen.

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