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- Materials and methods
Such low-nitrification savanna systems are of major interest. First, savannas represent 25% of terrestrial biomes (Solbrig & Young 1993) and are second to tropical forests in their contribution to terrestrial primary production (Atjay, Ketner & Duvigneaud 1987). They are also predominant in the social and economic environment of Africa: more than half the surface of the African continent is covered by savannas and savanna–forest associations (Menaut 1983; Solbrig 1993). Moreover, in Africa, savannas are associated with the regions of highest human population growth (Scholes & Walker 1993). Savanna grasses from the Andropogoneae supertribe are of economic interest, in particular for pastures. They are widely represented in Africa, and were introduced to South America where they became invasive in some areas (Baruch, Ludlow & Davis 1985; San José & Fariñas 1991; Baruch & Fernández 1993; Klink 1996).
Third, little is known about the possible control of plants on nitrification. Such control could provide plants with potential advantages in competition for N, and induce changes in ecosystem N balance. Because nitrate can be lost easily from the ecosystem (through leaching or denitrification), the ability of plants to inhibit nitrification could therefore be considered as an adaptive trait allowing them to bypass microbial processes that limit productivity.
Finally, from a global change point of view these humid savannas of West Africa are considered as non-emitting areas for NO and N2O as a result of their extremely low nitrification potential (Le Roux et al. 1995; Serça et al. 1998).
In the Lamto reserve (Côte d’Ivoire), the shrub savanna (covering almost 55% of the land surface and dominated by a perennial member of the Andropogoneae, Hyparrhenia diplandra (Hack.) Stapf) has been identified by several authors as a non-nitrifying ecosystem (De Rham 1973; Abbadie & Lensi 1990; Lensi et al. 1992). However, a recently discovered site of >15 ha, with similar vegetation, surprisingly exhibits 15- to 240-fold higher nitrification activity (Le Roux et al. 1995; Lata et al. 1999; Lata et al. 2000). A comparison between the nitrifying site (high-nitrification, HN site) and a non-nitrifying site (low-nitrification, LN site) gave us the opportunity to study in situ the mechanisms involved in the control of nitrification. Lata et al. (1999, 2000) found that while the two sites were similar in their soil physicochemical characteristics or the species composition of the grass cover, nitrification activity was positively or negatively correlated with root densities of H. diplandra populations in the HN and LN sites, respectively. The existence of distinct high- or low-nitrification sites in this ecosystem could result from an action of this particular grass at the population level.
Two possible mechanisms by which H. diplandra could alter the rate of nitrification can be hypothesized. The first is that the grass is directly involved in nitrification inhibition and that H. diplandra originating from the LN site can inhibit nitrification whereas the HN-site ecotype cannot. This inhibition could be due to an allelopathic effect of grasses on nitrifiers through root exudation of compounds that inhibit the activity of soil organisms (such as phenolic acids and tannins), the rate of exudation being generally linked to the size of plants (Prikryl & Vancura 1980). The second hypothesis postulates an indirect involvement of the grass in the inhibition of nitrification through an ability to compete with nitrifying bacteria for ammonium, and that the two H. diplandra populations (from LN and HN sites) exhibit different competitive abilities for ammonium uptake.
The first objective of this field study was to examine the extent to which H. diplandra individuals from LN and HN sites could influence nitrification. We designed in situ experimental plots with transplanted H. diplandra individuals collected from both high- and low-nitrifying soils, and planted in both high- and low-nitrifying soils. We measured the nitrifying enzyme activity (NEA), the ability of nitrifiers to oxidize ammonium to nitrate, which avoids the short-term variations induced by climate or other environmental factors.
The second objective of this field study was to examine if the variations in NEA could be related to the grass biomass at the individual level. In order to easily test this hypothesis non-destructively, we first searched for correlations between above- and below-ground biomass and several size indices of individual plants. Then we looked for correlations between NEA in nitrifying and non-nitrifying sites and one of these size indices: the diameter of H. diplandra tussocks. These measures were done to confirm and refine, at the individual level, in situ measures by Lata et al. (2000) who found some correlations between root densities and NEA.
Finally, to discuss these results in the broader context of the N cycle, we measured potential net C and N mineralization and denitrifying enzyme activity (DEA) to test if variations in NEA between the two nitrifying sites could be related to other N-cycle processes.
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- Materials and methods
Our study is the first in situ demonstration of biomass-dependent control of nitrification by grasses at the individual level. In the Lamto savanna, sites occur with contrasting nitrification patterns (high and low), and individuals from these sites have a different influence on nitrification. This shows that, in this case, such control can be at the plant ecotype level (Seliskar et al. 2002) as well as at the species level (Wedin & Tilman 1990; Knops et al. 2002).
The transplantation of individuals originating from HN or LN sites into HN or LN sites showed that there was a clear plant effect on nitrification. The LN plants decreased NEA in the HN site down to values found in the LN site, whereas HN plants restored the NEA in the LN site to values found in the HN site.
Our results are consistent with previous measures showing strong correlations between nitrification and root biomass (Lata et al. 2000). Two hypotheses were put forward in the Introduction to explain these results.
We found a negative correlation between the biomass of LN plants and NEA, which is consistent with both hypotheses. On the other hand, we found a positive relationship between plant biomass originating from the HN site and NEA. This may seem surprising due to the mostly autotrophic character of nitrification, so this process is generally considered not to be positively affected by root exudates (Bock, Koops & Harms 1989). However, the substrate for nitrification is ammonium produced by the heterotrophic mineralization of organic matter. Lamto savanna is highly structured, and micro-organisms and the production of organic and inorganic (such as ammonium) compounds are concentrated close to roots (Abbadie, Mariotti & Menaut 1992). Thus increases in plant biomass could potentially increase ammonium availability in the soil through an increase of root-derived carbon. Depending on the competitiveness for ammonium uptake between H. diplandra and soil micro-organisms, this could increase NEA with increasing plant biomass. Moreover, because nitrifiers can grow in mixotrophic or heterotrophic media (Bock, Sundermeyer-Klinger & Stackebrandt 1983), and survive and multiply in soils under heterotrophic conditions (Degrange, Lensi & Bardin 1997), the increase of nitrification with plant biomass could be caused by heterotrophic nitrification (Nemergut & Schmidt 2002). Our data cannot distinguish between the competition and the inhibition hypothesis. However, preliminary laboratory measurements on NEA in mixed HN + LN soils appear to favour the inhibition hypothesis, because the presence of a very low quantity of LN soil induces a drastic fall of NEA in HN soil (data not shown).
In conclusion, the mechanisms(s) involved in the inhibition of nitrification by H. diplandra remain unknown, but the hypothesis of an allelopathic inhibition through exudation of product(s) by the grass roots, as previously described (see Introduction), is still possible. However, only the identification of the (hypothetical) chemical mediator(s) responsible for inhibition can answer this question. Experiments based on in vitro evaluation of the influence of soil extracts from the two sites on pure culture of nitrifiers are currently under way.
From an evolutionary viewpoint, a major question remains. Is the HN site a remnant of a system where high nitrification was the rule, or is it a first indication of a change towards a nitrifying system? Both systems can coexist for a long time, as suggested by physiological adaptations (genetic differences in the inducibility of the assimilatory enzyme nitrate reductase between HN and LN H. diplandra populations; Lata et al. 1999). However, plants from the HN site are smaller, and grow more slowly in both glasshouse and field conditions, than those originating from the LN site (Lata 1999). The LN plants could therefore outcompete HN plants unless particular environmental conditions or perturbations maintain HN plants in pure communities in some areas. Thus, even if we cannot state which nitrification system appeared first, it appears very unlikely that Lamto savanna changes towards a high-nitrification system.
The ability to inhibit nitrification, either through an inhibiting factor or a superior competitiveness of plants compared with micro-organisms, could give a strong competitive advantage to LN plants. In acid soils it could create better local availability of N by decreasing its losses through nitrate leaching and denitrification. Carbon- and N-mineralization potentials were not significantly different between LN and HN sites; this suggests that the initial decomposibility of the organic matter is the same in the two systems, and that nitrification plays a key role. The DEA was ≈10-fold less in the LN site than the HN site, decreasing potential atmospheric losses of N.