The obvious way to assess the validity of the calculations given above is by measuring the amounts of N2fixed in the rhizosphere, but unfortunately this is not easy to do.
This subject has been thoroughly reviewed by Boddey and colleagues (Boddey, 1987;
Boddeyet al., 1995, 1998). Most studies to date have not differentiated whether N2
is being fixed endophytically or in the rhizosphere.
Proof of N2-fixation using15N2
The first question in the study of rhizosphere N2-fixation is simply to prove that it does take place. Uptake of15N2fixed by bacteria in the soil has been demonstrated in sugarcane (Ruschelet al., 1975), rice (Yoshida and Yoneyama, 1980), sorghum and millet (Gilleret al., 1988) and the tropical grassesDigitaria decumbensandP. notatum (De Polliet al., 1977). In all of these experiments the roots and soil were incubated in
enclosed chambers for short time-periods under controlled conditions. The results conclusively demonstrated that N2 had been fixed in the rooting medium and assimilated by the plant but did not distinguish whether the N2 was fixed in the rhizosphere or in soil not closely associated with the roots. However, in other experiments, fixation of15N2by bacteria was clearly shown to occur on or within excised roots of maize (Elaet al., 1982).
In some cases rapid uptake of the fixed N by the plants was found (within 72 h of exposure to15N2; Gilleret al., 1988) but the available results are contradictory. In one set of experiments with rice, uptake of newly fixed N was slow (Eskewet al., 1981) whilst other workers found 25% of the fixed15N in rice plants at the end of a 13-day incubation (Yoshida and Yoneyama, 1980). Comparison of15N2incorpora- tion with ARA estimates led Okonet al. (1983) to conclude that only 5% of the N2
fixed in the rhizosphere had been taken up bySetariaover 21 days, but this small recovery could be due to errors in the estimates of total N2-fixation rates, which were extrapolated from the ARA measurements.
These experiments give no evidence as to the mechanism by which N might be released from the bacteria. It is tempting to explain rapid uptake of fixed N by plants as being due to release of NH4 by living bacteria, but the death of bacteria and turnover of microbial N can also be extremely rapid. These experiments also cannot help in determining how much N2is fixed under field conditions. One attempt to measure N2-fixation associated with field-grown sugarcane using15N2resulted in an increase in15N-enrichment of the soil, but not in enrichment of 15N in the plant (Matsuiet al., 1981).
Estimates using the acetylene reduction assay
Inaccuracies in the use of ARA for measuring associative N2-fixation that result from the natural production of ethylene in soil have already been described (Chapter 4).
Unfortunately there are many other problems with this method. Early measurements were often made over long incubation periods of 24 h or more on cores of soil con- taining grass roots (e.g. Döbereineret al., 1972). It was found that there was a long lag phase of 12–24 h before significant activity was detected. This lag phase could be extended by the addition of fertilizer-N and only when all the N was used up were large rates of N2-fixation observed (Fig. 6.1). Thus the lag phase is almost certainly due to exhaustion of available N supplies prior to the induction of nitrogenase. Long incubation times were also made necessary by the slow rates of diffusion of C2H2and C2H4through compact cores of soil, and this exacerbated the problem of induction of artificially high rates of ARA as N deficiency led to artificially high induction of nitrogenase activity.
Various different methods of incubating grass roots under acetylene have been described. These include intact soil cores in the field, soil cores taken and incubated in containers, plants grown in containers in the glasshouse and roots excised from the plant (van Berkum and Bohlool, 1980). But all of these methods suffer from various errors and, even where immediate linear rates of ethylene production are detected,
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much of the ethylene may come from endogenous ethylene production (Sloger and van Berkum, 1988).
If all of these problems are considered – in addition to the fact that at best the acetylene reduction assay can only provide a spot measurement of nitrogenase activ- ity – then use of this method should be discouraged. Clark (1981) suggested that ‘the ready acceptance of erroneous values is due to our enthusiasms’ when considering ARA estimates of large amounts of N2-fixation associated with grasses. Despite the detailed critique of the method given by van Berkum and Bohlool (1980) and later comments (Boddey, 1987; Giller, 1987), some papers are still published without due reference to the methodological problems.
It must be recognized that there are many reports in the literature of high rates of ARA (> 1–2mM per plant h-1) associated with the roots of cereals and grasses that are in some cases comparable to rates found with nodulated legumes. These large ARA values are often extremely variable from plant to plant and are often only obtained in very moist conditions. Several research programmes to select genotypes of cereal crops for N2-fixation on the basis of ARA have been proposed – for instance, with rice (Ladhaet al., 1987) and with sorghum and millet (Waniet al., 1984). With sorghum and millet, very high (yet variable) rates of ARA were only found when the growth media were watered to 60–70% of their water-holding capacity and were maximal when organic manure was added to the growth medium. This latter treatment would almost certainly override the effects of the roots in stimulating N2-fixation by providing carbon substrates; in addition, the wet soil conditions required to promote high rates of ARA with sorghum and millet would rarely be experienced for long periods in the field.
With rice, consistent differences in root-associated ARA were found between genotypes (Ladhaet al., 1986) which were roughly proportional to the amount of plant biomass produced. Again, variability between plants was large (Tirol-Padre Fig. 6.1. The lag period before the onset of acetylene reduction activity associated with grass roots and the effect of added N fertilizer on the length of the lag phase. (Redrawn from van Berkum, 1978.)
et al., 1988) and controls to estimate endogenous ethylene produced in soil were not included. It is impossible to extrapolate from these ARA estimates to actual quantities of N2fixed. In another study the differences in ARA between genotypes were not correlated with variations in the natural abundance of15N (d15N) in grains of rice (Watanabeet al., 1987a). However, these authors point out that differences ind15N will depend on the efficiency of uptake of fixed N as well as on the rate of N2-fixation in the rhizosphere, which is what it is supposed that ARA indicates. Neither of the research establishments at which this work on sorghum, millet (ICRISAT) and rice (IRRI) was conducted is pursuing selection programmes for enhanced ARA at present.
N balance studies
Much of the early evidence used to support the case that large amounts of N2are fixed in association with grass roots came from N balance studies (Moore, 1966;
Dart, 1986). Several experiments were reported where different grass or cereal plots were shown to accumulate as much as 80 kg N ha-1even though legumes were absent (e.g. Nye, 1958; Jones, 1971). In short-term trials where a large amount of soil N is present, gains of less than 100 kg N ha-1can be difficult to measure (Dart and Day, 1975). Moreover, simple maintenance of cereal crop yields under continuous crop- ping cannot on its own be taken as evidence for gains from N2-fixation. It is not at all clear from such studies where the N is coming from and there are several possiblities for gains not due to N2-fixation. For example, although the contribution of N in rainfall may be small (< 5 kg N ha-1year-1) away from industrialized areas, uptake of N from deep soil layers may be important and might be overlooked in the N balance calculations. Gains of N could also, at least in part, be due to N2-fixation by cyanobacteria when rainfall is sufficient. Estimates for N2-fixation by cyanobacteria of 30 kg N ha-1have been made in temperate arable agriculture (Wittyet al., 1979), where temperatures are considerably lower than those prevailing in most tropical soils, and cyanobacteria are abundant in many tropical upland soils (Roger and Reynaud, 1982).
15N isotope dilution experiments
More recently the15N isotope-based techniques have been employed to estimate amounts of N2 fixed in the rhizosphere. The15N natural abundance method has been judged to be insufficiently sensitive to measure N2-fixation by free-living bacteria (Shearer and Kohl, 1988). Friedet al. (1983) concluded from experiments with legumes that the 15N isotope dilution method was unlikely to be sensitive enough to detect rates of N2-fixation of less than 10% of plant N. Nevertheless, it has been employed in this context.
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Limitations of the methods
The main limitation in the use of isotope dilution for the measurement of associative N2-fixation, apart from the issue of sensitivity, is the requirement for a non-fixing control plant. Comparisons of values derived from15N isotope dilution measure- ments with wheat genotypes grown in vermiculite, with or without inoculation with N2-fixing bacteria, gave indications of gains from N2-fixation (Rennie, 1980).
However, given the proliferation of root hairs and stimulation of nutrient uptake often caused by inoculation with N2-fixing bacteria, uninoculated plants are not likely to be good reference plants in such experiments. Moreover, later experiments showed that vermiculite can gradually release substantial amounts of unlabelled N (Gilleret al., 1986) so that differences in isotope dilution between inoculated and uninoculated plants may be due to the amount of unlabelled N they were able to exploit and not due to N2-fixation. These results highlight the danger of simply attributing N gains that are not accounted for to N2-fixation.
Glasshouse studies
Attempts to measure N2-fixation associated withL. fuscagrown in sterile soil that had been amended with15N and cellulose prior to the experiment were confounded, because the inoculated plants had roots with many more branches and root hairs (Maliket al., 1987). Separation of the soil organic N into different acid hydrolysable fractions showed an isotopic dilution in all of the different pools of N in soils inoculated with N2-fixing bacteria. The isotope dilution was greatest in the fraction that represented the N in the microbial biomass. In other experiments withL. fusca, another salt-tolerant grass (Polypogon monospeliensis) was found to be poorly matched as a reference plant and instead large fertilizer N additions were used to suppress any N2-fixation and to provide a non-fixing reference treatment (Malik and Bilal, 1988).
This approach may also introduce errors if the added fertilizer influences the availability of soil N in any way (Witty and Giller, 1991).
More recently the 15N isotope dilution method and the natural abundance methods have been employed to study N2-fixation with rice in flooded soils in the glasshouse. The two methods gave similar results, against a background of strong declines in15N enrichment of the available soil N, indicating that rice genotypes derived between 0 and 32% of their N from N2-fixation (Malarvizhi and Ladha, 1996; Shrestha and Ladha, 1996).
‘Field’-based estimates of N2-fixation
Most workers have selected grass species or genotypes of cereal crops that have small amounts of ARA for use as reference plants in isotope dilution experiments.
Several studies in Brazil have been conducted with different tropical grasses as test or reference plants in which the N-difference method and isotope dilution estimates have suggested similar amounts of N2-fixation. The batatais cultivar ofP. notatum, which hasA. paspaliin the rhizosphere, was estimated to gain 8–25% of its N from N2-fixation, equivalent to approximately 20 kg N ha-1 when compared with the cultivar pensacola (Boddeyet al., 1983). These studies were conducted in concrete cylinders filled with soil and sealed at the base with a cloth to restrict root growth but
allow drainage. Roots of the grasses did in fact penetrate into the soil below the cylinders, but the authors concluded that this was unlikely to introduce any significant error, as little N was present in the underlying soil.
Similar experiments were conducted with Paspalum and four other pasture grasses of the genusBrachiariain which different methods of adding15N either as a soluble fertilizer or as labelled organic matter were compared (Boddey and Victoria, 1986). The different grasses were shown to exploit N from different depths in the soil profile, and the15N-labelled organic matter gave more uniform15N-enrichment than
15N-labelled fertilizer and was thus chosen as the better method of labelling the soil N. The two grasses that were found to gain the largest proportion of N from N2-fixation were B. humidicola (30%) and B. decumbens (40%) but these were planted (later than the other grasses) in cylinders where soybean had been grown in a previous experiment.
Further experiments compared ecotypes ofPanicumfor N2-fixation using the residual labelling of15N in the same concrete cylinders that had been used for several other experiments and thus gave a stable enrichment of15N with time (Miranda et al., 1990). The proportion of N from fixation in thePanicumwas estimated to be 24–38%, equivalent to 5–10 kg N ha-1month-1 in the summer, usingBrachiaria arrecta(syn.B. radicans) as a reference plant. Miranda and Boddey (1987) compared benefits toPanicumecotypes from N2-fixation in pots amended with15N-labelled compost. The15N-enrichment of thePanicumecotypes decreased with time, whilst that of B. arrecta tended to increase (Fig. 6.2). Estimates of 16–36% of N from N2-fixation were obtained, which correlated roughly with total N yield. There was not a particularly good agreement in the ranking of N2-fixation between those genotypes included in both of their studies.
Much attention has focused on N2-fixation associated with sugarcane. Two sugarcane genotypes grown in cylinders of soil amended with15N-labelled organic
Fig. 6.2. Atom %15N of two ecotypes ofPanicum maximumand a single geno- type ofBrachiaria arrectagrown in15N-labelled soil. (After Miranda and Boddey, 1987.)
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matter were estimated to obtain 20–40% of their N from N2-fixation, using non-nodulating soybean as the reference crop, but the sugarcane did not accumulate significantly more N than the non-nodulating soybean (de Freitaset al., 1984). In a 2-year study with sugarcane grown in large pots containing 64 kg of soil labelled with
15N-urea, more than 60% of the N was estimated to have come from N2-fixation in one treatment, a conclusion supported by both15N isotope dilution and N balance methods (Limaet al., 1987). The data from this experiment were extremely variable, with coefficients of variation often close to 50%, and no mention was made of the prevention of growth of cyanobacteria. Further experiments were conducted in a large concrete tank filled with soil and carefully labelled with 15N fertilizer and organic matter, which was mixed throughout the soil (Urquiagaet al., 1992). Initial results were confounded by a declining 15N-enrichment of available soil N from around 1 atom %15N excess to 0.16 atom %15N excess over the first year. After 3 years this stabilized and estimates of 60–80% N from fixation (up to 270 kg N ha-1 year-1) were obtained, which were supported by N balance data. The plants were watered with tap water according to the soil moisture status, but no indication was given as to the nitrate concentrations in the water. Presumably the larger plants had a greater demand for water, and would therefore have received more nitrate. When rice was later sown in these tanks, no evidence for N2-fixation was found among 40 varieties (Boddeyet al., 1995).
The only true field study appears to be that of Yoneyamaet al. (1997), who compared the naturald15N abundance of sugarcane growing on farms in Brazil, the Philippines and Japan with that of neighbouring plants. Results were highly variable, indicating that between 0 and 76% of the N in sugarcane came from N2-fixation.
Can the results be accepted with confidence?
The attention to detail given in these experiments by Boddey and co-workers is extremely useful as it allows an evaluation of the possible flaws in the methods. There still remains a possibility that some of these differences between grasses in15N isotope dilution,d15N and N accumulation could be due to differences in their ability to access soil organic N, as Miranda and Boddey (1987) acknowledge, perhaps due to effects of the grasses on mineralization processes. However, the evidence to date supports the conclusion that the results are due to N2-fixation. This is an important area for clarification in the future – there is still no firm field information on the contribution of N from N2-fixation.