The first widespread agricultural exploitation of free-living heterotrophic N2-fixing bacteria was the sale of ‘Azotobacterin’ in Russia in the 1950s (Brown, 1974). Inter- est in this crop inoculant, which containedAzotobacter chroococcum, was short-lived as responses of crop growth of 10–20% were only found in some 30% of experimen- tal trials conducted. The most encouraging results were obtained with horticultural crops.
Interest in the use of free-living N2-fixing bacteria in agriculture gained a new lease of life with the discovery of ‘associative symbioses’ – the preferential occurrence of free-living N2-fixing bacteria in the rhizosphere of cereals and grasses (Döbereiner,
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1961, 1966; Döbereiner and Day, 1976). Detection of large amounts of ethylene when the new acetylene reduction assay was used with soil cores of tropical grasses (Döbereineret al., 1972) led to a period of intense and exciting research activity in this field. But it was the report that inoculation of grasses, and in particular cereal crops, withAzospirillumcould increase growth and yields (Smithet al., 1976) that concentrated much research effort on the goal of exploiting free-living bacteria to increase agricultural production.
It is worth noting that early research determined that the increases in plant growth observed on inoculation withAzotobacterwere caused not by N2-fixation but by bacterial production of plant growth hormones (Brown and Burlingham, 1968;
Barea and Brown, 1974). This research was conducted in the same department at Rothamsted Experimental Station, UK, as the initial experiments to measure the ARA associated with sugarcane andPaspalumroots (Döbereineret al., 1972). It was also soon realized that measurements of N2-fixation rates by ARA in the laboratory bore little relation to N2-fixation activity in the field (Witty, 1979; van Berkum, 1980). Despite this clear and long-standing evidence, reports still base evidence for associative N2-fixation on such methods. However, the largest published estimate of 2.4 kg N ha-1day-1fixed in association with maize roots, which was based on ARA measurements (von Bulow and Döbereiner, 1975), perhaps helped this field of research by attracting some healthy scepticism.
The following section will review the information now available on the occurrence and activity of heterotrophic N2-fixing bacteria in the rhizospheres of non-legume plants and attempt to draw some tentative conclusions about their importance in agriculture.
Associations between N2-fixing bacteria and roots of grasses The number of microorganisms is normally much greater in the rhizosphere than in soil further away from plant roots, due to the greater availability of carbon sources (Katznelsonet al., 1956). However, the amount of N available in the rhizosphere is often limiting for microbial growth (Stotzky and Norman, 1961) and so bacteria that can fix N2 would be expected to have a competitive advantage. In fact, most observations indicate that only 1–10% of the total bacterial population in the rhizosphere are N2-fixing bacteria (Okon, 1982; Patriquinet al., 1983). An exception is found in the case of rice, where up to 85% of the rhizosphere bacteria may be N2-fixers (Nayaket al., 1986). However, the accuracy of such counts is always doubtful, as they depend on the use of growth media that are selective for particular groups of organisms.
Specificity of the associations
A number of reports suggest that a degree of specificity exists between certain free- living N2-fixing bacteria and different grasses. Numbers ofBeijerinckiawere greater in the rhizosphere of sugarcane than that of other grasses and Beijerinckia was preferentially enriched in the rhizosphere population compared with the surrounding
soil population (Döbereiner, 1961).Azotobacter paspaliwas found in large numbers only in the rhizosphere of tetraploid cultivars of P. notatum (Döbereiner, 1966, 1970). Spirillum lipoferum was rediscovered and found to be widespread in the rhizospheres of tropical (Döbereiner and Day, 1976) and temperate grasses (Vlassak and Reynders, 1978). TheseSpirillumisolates were then classified in the new genus Azospirillumwith two species:A. lipoferumandA. brasilense(Tarrandet al., 1978).
A. brasilensewas found in large numbers on roots of wheat that had been surface sterilized in 1% chloramine-T, implying that the bacteria were so closely associated with the root that they were protected from surface sterilization (Table 6.1). In contrast, greater numbers of A. lipoferum were found on the roots of a different species, maize, when subjected to the same treatment (Baldani and Döbereiner, 1980).
Umali-Garciaet al. (1980) showed thatA. brasilensewas attached to root hairs of millet in greater numbers thanKlebsiellaorAzotobacter. Specific chemotaxis of Azospirillumstrains towards the roots of the hosts from which they had been isolated has been observed (Reinholdet al., 1985). A possible link between this chemotactic response and host root exudate was suggested. Strains from roots of maize or Kallar grass (Leptochloa fusca) were strongly attracted toL-malate, whereas a strain isolated from wheat roots was more strongly attracted to other organic acids abundant in exudates of wheat roots. A heat-labile compound of high molecular weight found in the exudates ofL. fuscaspecifically attracted theAzospirillumstrain isolated from this grass. Study of the rhizosphere flora of this salt-tolerant grass led to the identification of the new species, A. halopraeferens (Reinhold et al., 1987). A new genus of heterotrophic N2-fixing bacteria,Azoarcus(Reinhold-Hureket al., 1993), was also first isolated from the rhizosphere of Kallar grass, in which it also occurs as an endophyte (see below).
Do the bacteria invade living cells?
Although it was postulated in 1976 thatAzospirilluminvades live cortical cells of the root (Döbereiner and Day, 1976), this has not been confirmed. Matthewset al.
(1983) detected A. brasilense within pearl millet roots by immunochemistry but found no evidence for invasion of living plant cells. Most of the bacteria were present on the root surface or were found in intercellular spaces or in dead cells within the
Log no. g-1fresh wt % Inoculant strain
Site Control Inoc. Control Inoc.
Rhizosphere soil Washed roots
Surface sterilized roots
5.30 6.40 3.16
5.71 7.12 3.74
2.0 2.0 0 .
51 72 84 Table 6.1. Establishment of the inoculum strain Sp 245 ofAzospirillum brasilense in the rhizosphere and roots of field-grown wheat in Brazil. (From Baldaniet al., 1986b.)
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root where the epidermis was ruptured. Similar results were obtained forA. brasilense infection of wheat (Levanonyet al., 1989). N2-fixing bacteria were detected within the roots of Kallar grass using immuno-gold labelling techniques (Reinhold and Hurek, 1988). The bacteria appeared to enter the roots at lateral root junctions and were present between cells of the root cortex.
Is this a symbiosis?
Is there sufficient evidence of specific cooperation to allow definition of the associa- tion ofAzospirillum(or other genera of free-living N2-fixing bacteria) and the roots of grasses as a symbiosis? The evidence cited above certainly indicates that some species or strains of bacteria are better adapted to life in the rhizospheres of particular grasses. This is hardly surprising, as it is known that the chemical composition of root exudates varies substantially between species (see above) and the pH of the rhizosphere can differ markedly between genotypes of the same species (Brown and Bell, 1969). The evidence supports the suggestion that different strains and species of bacteria have evolved to occupy microenvironments provided in the rhizospheres of different crops. But this cannot be considered (as has been suggested in the past) to be equivalent to the legume–Rhizobiumsymbiosis. Whether the term ‘associative symbiosis’ is an appropriate description depends on how strict a definition is placed on the term symbiosis, but the use of the word associative is certainly accurate.
The term ‘diazotrophic rhizocoenoses’ (diazo= N2;trophic= nutrition;rhizo= root;
coeno= common) was once proposed to describe such associations (Vose and Ruschel, 1981) but fortunately was not generally adopted.
Root carbon as an energy source for N2-fixation
Carbon lost from living and dead roots, a process that has been called ‘rhizo- deposition’ (Whipps and Lynch, 1985), provides an important substrate for soil microorganisms. The amount of N2 fixed in the soil is limited by the amount of carbon available and the ability of the heterotrophic N2-fixing bacteria to capture and use it efficiently. Various calculations of the rates of N2-fixation that could be supported, depending on the amount of carbon available, have been made. These calculations are limited in accuracy by the difficulty of estimating how much carbon is translocated to the roots of plants, and of estimating how much of this is available for use by soil bacteria once root respiration and growth have been accounted for.
However, it is possible to gain an estimate of the maximum amounts of N2-fixation that could be sustained for a given input of carbon, assuming other factors are not limiting.
Estimates of the amount of carbon fixed from photosynthesis that is translocated below ground have been made for various temperate cereal crops and grasses. Values of up to 30% of total fixed carbon translocated for wheat, barley and maize (Barber and Martin, 1976; Helal and Sauerbeck, 1983; Swinnenet al., 1994a,b) and 50% in a grassland have been obtained (Warembourg and Paul, 1977), such values being equivalent to 1–3 t carbon ha-1(Whipps, 1990). No such estimates are available for
tropical cereals and grasses, and it is possible that the amounts of carbon available may be substantially greater in tropical grasses, with the more efficient C4- photosynthetic pathway. Of the carbon that is translocated below ground, up to half is respired directly by the plant (Swinnenet al., 1994a). Of the rest that does enter the soil and become available for use by microorganisms, little is present in soluble root exudates and the main addition is thought to be from death and decay of roots (Newman, 1985).
An estimate of the amount of N2-fixation that might be supported by this carbon can be made by considering the composition and metabolic capabilities of rhizosphere microorganisms. None of the free-living N2-fixing bacteria studied can use polysaccharides or other more complex carbon compounds for growth (Brown, 1982). The fixation of N2in the rhizosphere will thus depend on the ability of the N2-fixing bacteria to capture and use either simple carbon compounds in root exudates or the breakdown products of more complex root carbohydrates provided by other microorganisms. The efficiency of carbon utilization for N2-fixation by pure laboratory cultures of heterotrophic N2-fixing bacteria ranges from 4 to 174 g C g-1 N2-fixed in the laboratory (Giller and Day, 1985) and may be much less under field conditions. Therefore, it is assumed that the efficiency of use of root carbon in N2-fixation is 10 g C g-1 N2-fixed (a rather generous estimate), that the N2-fixers comprise 10% of the total bacterial population in the rhizosphere and that they can acquire carbon in the rhizosphere in proportion to their numbers, then for every 100 kg C ha-1that is translocated below ground, 1 kg N ha-1will be fixed. The greatest flaw in such a calculation is that quantities of carbon available for N2-fixation cannot be estimated accurately. This is because, as indicated already, most of the heterotrophic N2-fixers cannot utilize complex carbon compounds and thus only a small proportion of the available carbon is likely to be used directly for N2-fixation.
From such calculations it seems likely that the true amount of N2-fixed in the rhizosphere may be closer to 1 kg N fixed for every 1000 kg C translocated below ground.