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In recent years, there has been increasing recognition of the need for a broader perspective for plant ecology that incorporates interactions with soil microbial communities (Reynolds et al. 2003; Schimel et al. 2007). A range of studies have shown that plant–soil interactions and the dynamic feedbacks these interactions generate, can be important organizing forces of community structure and function through both direct and indirect pathways (Klironomos 2002; Bever 2002a,b, 2003). At the same time, plant and soil communities are themselves directly influenced by environmental factors such as soil pH and salinity levels, soil toxicity and water availability. Particularly for associations between plants and symbiotic mutualists, quantifying how variation in environmental factors influences the effectiveness of the interaction is central to understanding the dynamics of community assembly (Thrall et al. 2007a), as well as how these associations co-evolve across geographic ranges (Parker 1999; Thompson 2005). In this context, the presence of trade-offs in different life-history components (e.g. N2-fixing effectiveness vs. growth rate or tolerance of hostile soil factors) could strongly determine the ecological outcomes of evolutionary processes.
There are several potential mechanisms through which evolutionary shifts in host plant interactions with soil symbionts could mediate plant adaptation to productivity or stress. First, it is possible that plant dependence on symbionts is unchanged across an environmental gradient, but that the micro-organisms themselves are sensitive to this gradient. In this case, microbial adaptation to the environmental stressor is a prerequisite for plant success and the presence of trade-offs in different microbial life-history components (e.g. growth rate vs. tolerance of hostile soil factors) would underlie the microbial evolutionary dynamic. There is evidence of such trade-offs and of the resulting adaptation of soil micro-organisms to stress (Schimel et al. 2007). For soil symbionts, there is also some empirical support for this adaptation resulting in improved plant performance in more stressful environments. For example, Toler et al. (2005) showed that Sorghum bicolor performed better in the presence of heavy metals in association with metal tolerant strains of mycorrhizal fungi, and Stahl & Smith (1984) found that Agropyron smithii exhibited greater drought tolerance when grown with mycorrhizal fungi isolated from arid environments compared to isolates from more mesic environments in the western United States.
Similarly, there is at least some evidence that the ability of legume hosts to grow and survive in saline conditions is improved when they are inoculated with salt tolerant strains of rhizobia (Zou et al. 1995; Hashem et al. 1998; Shamseldin & Werner 2005). In contrast, Lal & Khanna (1994) found no evidence of a benefit of rhizobial salt tolerance for plant performance. Moreover, it has been shown in some cases that microbial evolution in response to environmental gradients can lead to a decrease in mutualistic benefits (Corkidi et al. 2002; Kiers et al. 2002) and there is theoretical support for this (Thrall et al. 2007a). Overall, given conflicting results and the relatively limited number of symbiont strains evaluated in most of these studies, these examples must be regarded as illustrative, leaving the generality of these findings still undetermined.
It is also possible that plant dependence on soil symbionts might shift along an environmental gradient (e.g. in more stressful environments, hosts might evolve reduced dependence on mutualists because of associated costs or trade-offs relevant to other aspects of persistence). Plants, for example, are known to vary in dependence on mycorrhizal fungi and this variation is thought to relate to the ecology of individual plant species. Plants with low dependence on mycorrhizal fungi tend to dominate in areas with low densities of mycorrhizal fungi or where soils contain high nutrient levels (e.g. Medve 1984). Consistent with this mechanism, Schultz et al. (2001) found that ecotypes of big bluestem (Andropogon gerardii Vitman) from fertile prairies are not dependent on mycorrhizal fungi, while ecotypes of big bluestem from infertile prairies are highly dependent.
A third possibility is that adaptation and persistence along an environmental gradient requires genetic change in both the plant and symbiont. In this case, the specificity of the interaction may result in plant genotypes doing best with the symbiont genotypes from their same environment (i.e. co-adaptation). Necessary elements of this process are well-established, including genetic changes along environmental gradients in relation to the symbiosis in the host (Schultz et al. 2001) and symbiont populations (Ibekwe et al. 1997; Delormea et al. 2003). There is also abundant evidence of specificity in host response to symbionts (Burdon et al. 1999; Thrall et al. 2000; Bever 2002a; Klironomos 2003) and in symbiont response to hosts (Bever 2002b). Tests in homogeneous environments have demonstrated negative feedback, which deteriorate mutualisms rather than lead to co-adaptation (Bever 2002a; Castelli & Casper 2003). However, evolution along an environmental gradient may be more likely to yield positive feedback that would generate co-adaptation (Bever 1999). Co-adaptation of host–symbiont associations along environmental gradients has yet to be conclusively demonstrated, in that (as far as we are aware) there are no studies which provide evidence that matching of adapted plant and symbiont populations results in synergistic advantages in performance.
Soil salinity is one example of an environmental stress which has become an issue of major ecological and economic importance in many parts of the world. For example, in Australia the extensive clearing of deep-rooted native perennial vegetation has resulted in significant increases in dryland salinity (National Land & Water Audit 2001). Given the prevalence and diversity of native shrubby legumes (e.g. Acacia spp.) in many Australian ecosystems (Groves 1994) as well as the nutrient-poor status of many of these soils, the association between these plants and N2-fixing rhizobial bacteria represents a model system for investigating ecological and evolutionary interactions along an environmental gradient such as soil salinity, and exploring how co-evolutionary shifts in these interactions might influence community structure and function.
Previous studies of Acacia–rhizobial associations have shown variation in abundance and diversity (Lafay & Burdon 1998, 2001), host specificity (Burdon et al. 1999; Thrall et al. 2000), and N2-fixing effectiveness in relation to host species, soil chemistry and physical environmental factors (Thrall et al. 2007b). Furthermore, it is known that both hosts (Marcar & Crawford 2004) and rhizobia (Elsheikh 1998; Zahran 1999; P.H. Thrall & L.M. Broadhurst, unpublished data) vary in salt tolerance. Of particular interest in the context of environmental stress is the identification of any underlying physiological trade-offs in key life-history features that might influence ecological and evolutionary trajectories. However, there has been little or no examination of this issue in natural plant–microbe systems, although such trade-offs could clearly impact on community composition and host–symbiont co-evolutionary dynamics.
We still lack a broad understanding of the causal links between environmental heterogeneity, population and geographic genetic variation, and ecological performance of host–symbiont associations. Improved knowledge of these interactions will not only provide insights into community and co-evolutionary dynamics, but it has applied value in the context of large-scale revegetation and ecosystem restoration of degraded agricultural landscapes (Thrall et al. 2005). Here we use a range of Acacia spp. known to vary in their ability to tolerate saline soils and associated rhizobial symbionts to address several basic ecological questions relevant to understanding the impact of an environmental stress factor (soil salinity) on a naturally occurring symbiotic association: (i) Is there evidence for a trade-off between rhizobial growth rate and adaptation to salt stress? (ii) Is there evidence that such a trade-off could mediate bacterial N2-fixing effectiveness and thus the ability to promote plant growth? (iii) Is there evidence that the relative performance of plant–rhizobia combinations varies along salinity gradients in relation to salt tolerance in either the host or the symbiont?
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This study affirms several basic tenets of microbial mediation of plant adaptation to salinity stress. First, we found a strong trade-off between tolerance to salt and growth rate in the absence of salt in rhizobia isolates. However, salt-tolerant strains of rhizobia did not differ from salt-sensitive strains in their ability to promote growth of a range of Acacia spp. even in saline soils. Moreover, while we did find strong evidence of host-specificity in the response of different Acacia spp. to particular rhizobial strains, we did not find that this specificity of response was aligned as expected under the hypothesis of co-adaptation. Overall, there was evidence of rhizobial mediation of Acacia adaptation to salinity, as we observed differences in salinity tolerance among Acacia spp. and salt-tolerant acacias differed from salt-sensitive species in their interactions with rhizobia, in that salt-tolerant acacias had reduced dependence on rhizobia. This reduced dependence could be a critical aspect of ecological success in highly saline conditions. We discuss the significance of each of these results in turn below.
First, our results show that strains of rhizobial bacteria with high tolerance to salinity also have reduced growth rates in the absence of salinity, as would be expected from a cost of tolerance to salinity (and has been shown more generally for a number of other environmental stress factors; Schimel et al. 2007). This result contrasts with the frequent conclusion that rhizobial growth rate and salt-tolerance are positively correlated (e.g. Elsheikh 1998; Marsudi et al. 1999; Barboza et al. 2000; Zerhari et al. 2000), although this difference may be attributable to the fact that most previous studies did not factor out intrinsic variation in growth rates from analyses of salt-tolerance. From a community perspective, the trade-off that we demonstrate suggests that salt-tolerant strains will be replaced by faster-growing salt-sensitive strains in soils of low salinity. That is, as a result of this cost, we expect to find genetic and species differentiation of rhizobial communities along a salinity gradient. Although not conclusive, evidence from other work on rhizobial salt tolerance (P.H. Thrall & L.M. Broadhurst, unpublished data) is consistent with this idea. In that study, RFLP analysis of strains from both non-saline and saline soils found that the majority of rhizobial genomic species present in saline soils were not present in the non-saline sites. Other studies have suggested that the relative predominance of different rhizobial genera may shift in relation to environmental stress (e.g. Barnett & Catt 1991; Jenkins 2003). More generally, soil microbial abundance, community structure and functional representation by different microbial groups have also been shown to respond to gradients in soil salinity (Omar et al. 1994; Nelson & Mele 2007; Schimel et al. 2007).
Clearly, shifts in symbiont community composition along environmental gradients could have consequences for the relative ecological success of different host species. Such shifts could also influence the co-evolutionary dynamics of host–symbiont associations, particularly where host specificity and symbiotic effectiveness are factors. In the case of Acacia–rhizobial interactions, previous studies have indeed shown considerable variation among host species in their responses to particular rhizobial isolates (Burdon et al. 1999; Thrall et al. 2000; Murray et al. 2001). We also found substantial variation in average growth promotion of rhizobial isolates and in the specificity of this growth promotion in the glasshouse. Depending on how rhizobial community composition responds to increasing salinity, and further how trade-offs in growth and tolerance relate to the levels of mutualistic benefits conferred, this could alter the relative competitive abilities of salt-sensitive vs. more salt-tolerant acacias along salinity gradients. In the context of co-adaptation, this could also lead to distinctly different co-evolutionary dynamics in different soils.
However, our current results do not provide evidence for this specificity of rhizobial growth promotion playing an important role in acacia growth in saline soils. In fact, we did not observe any differences in average growth promotion or specificity of growth promotion in a survey of 40 rhizobial strains chosen from across the salt-tolerance spectrum identified in the laboratory cultural experiments. Similarly, we did not detect any difference in growth promotion between the mixed salt tolerant inoculum and the mixed salt sensitive inoculum in the direct test of co-adaptation (glasshouse experiment II). Several previous studies, largely based on comparison of single pairs of rhizobial strains varying in salt-tolerance, have either shown that inoculation with salt-tolerant strains of bacteria may reduce the adverse impacts of salt relative to salt-sensitive rhizobia (Zou et al. 1995; Hashem et al. 1998; Shamseldin & Werner 2005), or found no added benefit of rhizobial salt-tolerance for plant performance in saline soils (Lal & Khanna 1994). Therefore, at present there is no consistent evidence that rhizobial adaptation to salinity generally benefits plant growth – the present study is perhaps the most comprehensive evaluation of variation in salt-tolerance in native systems to date.
Interestingly, we found that salt-tolerant species of Acacia had reduced responsiveness to rhizobia, both in survival and growth (the former may be an artefact, given that a positive response to N was constrained by high survival in the N-free control). In fact, A. salicina, which, along with A. stenophylla, exhibited very high levels of tolerance to salt (Fig. 2), did not benefit from rhizobial inoculation in any salt concentration (Fig. 6). Moreover, hosts with low salt-tolerance, such as A. mearnsii, had high responsiveness to rhizobial inoculation which was sharply reduced with increasing salinity. The reduced dependence of salt-tolerant acacias on rhizobia could be an adaptive response to several physiological and ecological forces (Fig. 7).
Figure 7. Conceptual diagram illustrating hypotheses regarding the relationships between soil salinity, rhizobial community composition and abundance, and impacts on the evolution of the plant–rhizobial interaction towards lower host dependence on the symbiosis in more saline environments (Δ = non-directional change). This could either be an indirect consequence of reduced rhizobial density (path B), a response to reduced physiological benefits from the association (path C), or changes in plant uptake of N (path A). Solid arrows indicate pathways for which there is empirical evidence, either from the current study or in existing literature, while dotted or dashed arrows indicate hypothesized relationships. Our study demonstrates a cost to rhizobia for salt tolerance which would generate genetic differentiation along a salt gradient. Our results also indicate a general decline in plant growth promotion (N2-fixing effectiveness) with increasing salinity, but do not support the idea that more salt-tolerant rhizobia differ in mutualistic benefits from salt-sensitive rhizobia (path D).
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First, there could be a reduction in the physiological efficiency of symbiotic N2-fixation associated with saline soils (identified as causal pathway C in Fig. 7). Consistent with this, we observed that across all Acacia spp. the responsiveness to rhizobia decreased with increasing salinity (Fig. 4b), while the responsiveness to N increased (in terms of both survival and growth). This suggests that while N limitation is still important in saline conditions, N-fixation becomes a less effective substitute for soil N. For example, N2-fixing activity has been shown to decline under salt stress (Zahran 1991, 1999; Lal & Khanna 1994; Hashem et al. 1998).
It is also possible that this reduction in efficiency is partly due to reduced rates of proliferation of rhizobial bacteria in saline conditions (causal pathway B in Fig. 7). A range of surveys have found that the overall density of rhizobia can be strongly reduced in saline soils (Singleton et al. 1982; Elsheikh 1998; Zahran 1999; Slattery et al. 2001). Impairment of the infection process by salinity has also been observed previously (Singleton et al. 1982; Craig et al. 1991; Zahran 1999) and our finding of reduced nodule density and poorer distribution of nodules with increasing salinity is consistent with this interpretation. The reduced density of rhizobial soil populations and the reduced efficiency of the association could alter the cost-benefit ratio in favour of reduced dependence as part of host adaptation to saline soils.
Another potential selective force for reduced dependence would arise if salt-tolerant rhizobia are less effective mutualists (causal pathway D in Fig. 7). This possibility is suggested by previous work (Zahran 1999), but is not supported by the present study. Finally, from an ecological perspective, cost benefit considerations of symbiotic N2-fixation also depend upon the availability of soil N, which might actually increase in high salinity due either to reduced interspecific competition and lower plant densities, or to negative impacts of salinity on nutrient uptake rates by plants (possibly due to reduced photosynthetic rates and demand for N; Sprent & Zahran 1988). This would also select for reduced host dependence on rhizobia (causal pathway A in Fig. 7). Relatively little work has examined nutrient dynamics in natural plant communities subject to soil salinity – one study of native Acacia species in the Negev desert found a positive relationship between soil salinity and nitrogen (and a negative relationship between N and plant diversity; Munzergova & Ward 2002), and strong positive correlations have been found between plant N and soil salinity in at least some agroecosystems (van Groenigen & van Kessel 2002). The potentially complex interactions between soil N, microbial activity, and plant community structure and function in relation to environmental stresses such as salinity are clearly worth further investigation in the current context.
A general expectation, based on both theoretical and empirical studies, is that mutualisms should be stronger in lower quality environments (Thrall et al. 2007a). There is good empirical support for this idea (e.g. the relationship between nitrogen fertilization and other aspects of soil fertility and the level of benefits conferred by rhizobia and mycorrhizal fungi: Johnson 1993; Corkidi et al. 2002; Denison & Kiers 2004). There is also support for shifts in host dependence on mutualists along such productivity gradients, for example, big bluestem (Andropogon gerardii; a dominant grass in central USA), derived from infertile sites, is highly responsive to mycorrhizal fungi, whereas the responsiveness of big bluestem from fertile sites is reduced (Schultz et al. 2001). The results from the current study appear to contradict this idea, in that more salt-tolerant Acacia species (presumably found in more saline, lower quality environments) actually showed less dependency on rhizobia than salt-sensitive hosts from less stressful environments. However, much of the theory on the evolution of host–symbiont interactions (Thrall et al. 2007a) assumes that the productivity axes along which host–symbionts evolve are also correlated with the axes relating to the symbiosis itself (e.g. gradients in N availability and legume-rhizobial mutualisms). This will clearly not be the case for many other factors relating to environmental quality. For example, in the current study the gradient in soil salinity is not closely correlated with a gradient in the mutualistic benefit of N2-fixation. This leaves the interaction free to evolve in other directions (e.g. reduction in host dependence in lower quality environments and possible increase in saprophytic ability/parasitism in the symbiont, rather than increased mutualism). This suggests that the evolutionary flexibility of host–symbiont systems depends on the degree of independence of the axes associated with the symbiosis vs. those associated with environmental quality.
Overall, the present study presents evidence for reduced strength of the plant–rhizobial mutualism as part of adaptation to salinity. Further work is necessary to separate the multiple causal pathways that could generate this result (Fig. 7). Given that Australian Acacia spp. also associate with mycorrhizal fungi (Warcup 1980), it would be of interest to examine how these might further modify plant responses to environmental stress. While shifts in nutritional mutualisms are expected along a fertility gradient, we do not know the extent to which shifts in dependence on microbial symbionts underlies plant adaptation to other environmental factors.