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- Materials and methods
The roots of the majority of plant species found in temperate grasslands form symbiotic arbuscular mycorrhizal (AM) associations, and AM fungi are the main mutualistic organisms associated with grasses (Newsham & Watkinson 1998). The mycelial systems produced by AM fungi form extensive networks, with hyphal lengths reaching up to several m cm−3 soil volume in undisturbed temperate grassland soil (Miller, Reinhardt & Jastrow 1995). These provide the major nutrient-absorbing interface between plants and soil, and form important pathways for the rapid transport of nutrients to plant roots (Smith & Read 1997). Increased plant growth following root colonization by AM fungi is normally due to an improvement in mineral acquisition. This is achieved by external hyphal uptake of mineral nutrients from outside the rhizosphere, followed by transport of these nutrients to the plant root. Phosphorus is, at least quantitatively, the most important nutrient taken up via AM hyphae (George, Marschner & Jakobsen 1995). Not only do mycorrhizal plants acquire more nutrients, it appears that they are able to share them via an underground network of hyphal connections linking individuals within and between species, indicating the existence of a ‘mycorrhizal web’ for the exploitation and redistribution of resources within plant communities (Grime et al. 1987; Simard et al. 1997, 2002; Newsham & Watkinson 1998; Wilkinson 1998; Hart & Klironomos 2002). If mycorrhizal fungi transfer significant amounts of nutrients among neighbouring plants, they may potentially act as significant integrators of plant community dynamics, affecting seedling establishment near mature plants, and resource availability for allocation to growth, reproduction, competition, defence, regrowth following defoliation, and other functions. Mycorrhizal-mediated resource movement among neighbouring plants may also strongly influence patterns of local neighbourhood competition, the rate and patterns of succession, and the maintenance of species diversity in plant communities (Grime et al. 1987; Eissenstat & Newman 1990; Miller & Allen 1992).
However, the precise role and importance of interplant hyphal nutrient and C transfer remain a matter of debate. It is clear that the relatively low level of host specificity of AM fungi permits both intra- and interspecific connections to form among neighbouring plants, and therefore common mycelial networks (CMN) could provide a direct mechanism for the transfer of reduced C, mineral nutrients, or other substances between plants. Grime et al. (1987) attributed increases in plant species diversity to extensive mycelial networks facilitating the direct flow of C or nutrients from sufficient to deficient plants. They suggested that the export of assimilate from ‘source’ (canopy dominants) to ‘sink’ (understorey components) through a CMN may be an important mechanism in maintaining species-rich communities. There is considerable recent evidence in support of this source–sink regulation of interplant nutrient (nitrogen, P) flux (for reviews see Simard et al. 2002; He, Critchley & Bledsoe 2003). However, mycorrhizal-mediated nutrient transfer may increase competitive dominance, as larger plants acquire greater resources from common mycorrhizal mycelium because of their greater nutrient demand (Zabinski, Quinn & Callaway 2002). Competitive intensity within plant communities may be affected simply by more diverse or different mycorrhizal associations of plants tapping into common networks, resulting in increased nutrient access for some plant species (Simard & Durall 2004).
The benefit of AM hyphal-mediated transport of C or nutrients to the host plant has been questioned. Fitter et al. (1998); Pfeffer et al. (2004) found that C was transferred between plants via AM fungal networks, but the C remained in the roots of the receiver plants. Fitter et al. (1998) interpreted this from a ‘mycocentric’ viewpoint: transfer through the mycelial fungus moves resources to parts of the mycelium where storage function is predominant, and separate from where C acquisition is occurring. Therefore their study provided no evidence that the translocation of C benefited the receiver plant. Likewise, whether P transport provides benefit to the host plant remains under investigation. Phosphorus transfer has been reported to be too slow or too small to affect the receiver plant's nutrient status significantly (Ikram, Jensen & Jakobsen 1994). Simard et al. (2002) suggest that the magnitude and rate of P transfer may be too small to be of ecological importance.
Alternatively, in some systems it appears that interplant transfer of P may be an important mechanism, allowing species a competitive advantage over less-mycotrophic neighbours. Zabinski et al. (2002) reported that mycorrhizal hyphal links allowed for increased interplant hyphal transfer of P and increased competitive ability of an invasive forb. Other studies have indicated that interplant transfer may make a significant contribution to the total P acquired by a plant. A glasshouse dual-pot and dual-labelling (32P and 33P) experiment estimated that a non-trivial amount (average 17%) of the total P taken up by a warm-season tallgrass prairie grass was acquired directly from its neighbour via hyphal-mediated transfer (Hartnett & Wilson 2002). Likewise, Martins & Read (1996) found that the majority of 32P isotope transfer between two forb species occurred by a direct pathway via AM mycelium; and Tuffen, Eason & Scullion (2002) found that common mycelial connections increased 32P transfer to receiver plants by approximately one-third.
It also remains unclear whether interplant transfers of resources occur over sufficient spatial scales to influence the relative performance and competitive abilities of neighbouring plant species, and whether a net movement of resources occurs towards particular plant species. Furthermore, if a net movement of resources via AM hyphae does occur, it remains unclear whether net flows move from resource-rich plants to relatively poor ones, or whether net flows increase the equitable distribution of limiting resources. A previous study in tallgrass prairie demonstrated the occurrence of non-random patterns of interplant 32P transfer among neighbouring species (Fischer Walter et al. 1996). That study showed that there was strong differential P movement to certain plant species, with non-equitable distribution of limiting nutrients among co-occurring species in tallgrass prairie. However, in that field study it was not possible to separate the effects of differential rhizosphere overlap and potential mycorrhizal hyphal transfer, thus it could not be determined if mycorrhizal mycelia were a mechanism for this transfer.
In this controlled glasshouse study, we assessed 32P transfer from a ‘donor’ plant to a ‘receiver’ plant of the same or different species. The potential contribution of mycorrhizal-mediated transfer was determined by growing plants together in containers and using 43-µm nylon root barriers to separate the plant rhizospheres of different individuals. This barrier allowed the penetration of hyphae between the rooting space of neighbouring individuals, but excluded direct contact of plant roots, eliminating differential rhizosphere overlap. An additional treatment was included in which fungicide was applied to suppress mycorrhizal transfer of nutrients, allowing for estimation of nutrient uptake via roots only. This study was designed to broaden our mechanistic understanding of the patterns and controls of interplant nutrient flow by quantifying P transfer between plants of different species, as well as of the same species.
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- Materials and methods
Comparisons between the ‘no barrier + fungicide’ treatment with the ‘43-µm-barrier – no fungicide’ treatment allows rough estimates of the proportional uptake attributable to root- vs hyphal-mediated transfer for these two prairie species. However, the probability of nutrient exchange, through both roots and AM hyphae, is much greater for plants with roots intermingled, compared with exchange that can occur only across the relatively small surface of the mesh barrier. Therefore the amount of nutrient transfer attributable to AM hyphae is certainly an underestimate. Even so, when these treatments are compared, nutrient acquisition attributable to AM-mediated transfer was >50% of the total 32P uptake for S. nutans, regardless of the donor species, and <20% of the total for A. ludoviciana. Conversely, isotope acquisition attributable to root uptake (loss from donor roots and subsequent uptake via receiver roots) was greater in A. ludoviciana than S. nutans. Therefore, while the dominant tallgrass prairie grass was highly dependent on AM hyphal transfer of nutrients, the subordinate forb appears to be more dependent on root uptake for P acquisition. In fact, direct root uptake by this forb made a significantly greater contribution to total plant P acquisition than did AM hyphal-mediated transfer.
The results of this study show that active AM colonization (no fungicide application) led to 32P transfer from donor to receiver plants. The lack of 32P in the receiver plants that were separated by the mesh barrier and received applications of fungicide indicated that isotope transfer through a soil pathway, involving leakage and subsequent uptake by roots adjacent to the other side of the mesh barrier, or diffusion of the isotope through the mesh, was negligible. This indicates that, when plants were separated by the mesh barrier with active mycorrhizae, P was transferred via a direct pathway through common mycelial interconnections, and/or exudates from donor-associated mycelium were immediately taken up by receiver-associated mycelium and transferred to the receiver plant. In either scenario, it is clear that uptake into receiver plants was primarily due to mycorrhizal-mediated transfer.
Several previous studies have examined the potential effects of mycorrhizae and common mycorrhizal networks on intra- and interspecific competition among plants, and all have suffered from inadequacies in clearly demonstrating the existence of a functional CMN (Leake et al. 2004; Simard & Durall 2004). Loss of nutrients from roots or hyphae into the soil pool, followed by immediate uptake by mycorrhizal hyphae, appears to be plant-to-plant transfer through a CMN. However, the technical problems in demonstrating unequivocally that plant-to-plant C or nutrient (P, N) transfer occurs via a CMN, ensuring that transfer is genuinely through hyphal interconnections, are formidable (Robinson & Fitter 1999; He et al. 2003). The linking of mycelial location, identification and function in soil remains a major challenge, but is beginning to be achieved via molecular identification of fungal species from roots and soil (Wallander et al. 2003; Leake et al. 2004). While our current study does not show conclusively whether CMN act as a conduit for direct nutrient transfer, it clearly demonstrates that mycorrhizae facilitated transfer. Transfer may have occurred through a common conduit of CMN, or exudates from roots or hyphae may have been taken up efficiently from the soil pool by mycorrhizal hyphae of the adjacent plant root. Either mechanism could result in changes in plant–neighbour interactions and community structure. It is clear from our data that nutrient transfer via mycorrhizal mycelium is not equitable between neighbouring species. Interplant transfer via AM fungi accounted for >50% of the total 32P acquisition by S. nutans, but accounted for only 20% of the 32P uptake into A. ludoviciana. This increase in P transfer between the conspecific grasses may be partially due to their higher mycorrhizal colonization levels compared with the subordinate forb. This increase in hyphal development may lead to an increase in nutrient absorption, and may also increase the potential for interplant hyphal connections. The increase in colonization may be due to the coarser root morphology of the grass (personal observation). Relationships between AM symbiosis and rooting patterns are well established as both are key characteristics that influence the acquisition of limiting soil resources. Frequently there is an increase in hyphal development as roots become coarser (Miller et al. 1995), and AM symbiosis may influence plant root architecture (Hetrick, Wilson & Leslie 1991). Warm-season grasses are typically highly colonized, and their colonization frequently exceeds that of co-occurring forbs (Wilson & Hartnett 1998).
The host-specificity of dominant AM fungi may also play an important role in determining the probability that a host plant is able to participate in a CMN. There is now increasing evidence that some mycorrhizal fungi are host-specific. Species of fungi may vary with host plants (Sanders & Fitter 1992; Bever, Westover & Antonovics 1997; Eom, Hartnett & Wilson 2000; Vandenkoornhuyse et al. 2003), and mycorrhizal species may differ significantly in their influence on plant growth and development (Hart & Reader 2002). For example, AM fungal species have been reported to differ in their capacity to transport P (Jakobsen et al. 2001; Smith, Smith & Jakobsen 2003, 2004). Recent studies found that the frequency of anastomoses per hyphal contact ranged from 64 to 78% (Giovannetti et al. 2001), but the formation of these mycelial networks depends on self-recognition between compatible hyphae, with hyphae of one morphospecies showing the ability to discriminate itself from others (Giovannetti 2001; Giovannetti & Sbrana 2001). Shifts in fungal species associated with the different plant species would be expected to reduce the potential to form CMN, possibly ensuring that the benefits of linking to a CMN are not shared too widely and preferentially benefit co-linked plants of the same species (Leake et al. 2004). The significantly greater transfer of hyphal-mediated 32P in S. nutans, compared with A. ludoviciana, may reflect an association with a different suite of AM fungal species. This, combined with the higher abundance of host-specific fungal hyphae, leading to greater opportunities for anastomoses between common mycelial strands, may be the mechanism explaining the increased nutrient transport between S. nutans plants, possibly to the exclusion of the subordinate forb.
Although there is a degree of specificity in mycorrhizal associations, there are AM fungi that are not selective as to host plant, and most AM fungal species can colonize most plant roots. In turn, most plants host several different AM fungal species concurrently (Merryweather & Fitter 1998). Indeed, AM fungal hyphae have been observed extending from roots of one plant species to another (Giovannetti 2001). In our study, hyphal-mediated P transfer was observed between species, regardless of donor plant, although this transfer was lower than grass-to-grass transfer. Even our conservative estimates indicated that ≈20% of the total P transferred to A. ludoviciana was attributable to hyphal–mediate transfer. This indicates either the presence of CMN through fungi associated with both hosts, or an extremely efficient system in which limiting nutrients are immediately taken up by neighbouring hyphae following release into the soil pool.
Several researchers have assumed that non-random nutrient sharing through mycorrhizal networks aids in the redistribution of resource equitability among neighbours within plant communities, thereby decreasing interspecific differences in resource acquisition and competitive ability, and thus contributing to coexistence and the maintenance of plant community diversity (Grime et al. 1987; Read 1997; van der Heijden et al. 1998; Giovannetti 2001; Simard et al. 2002). Several studies provide supporting evidence for reduced competition and increased diversity where plants are inoculated with mycorrhizal fungi (Grime et al. 1987; Gange, Brown & Sinclair 1993; van der Heijden et al. 1998). However, experiments in C4 grasslands indicate that mycorrhizal symbiosis decreases plant species diversity by increasing the competitive success and abundance of the dominant grasses relative to subordinate species (Wilson & Hartnett 1998; Hartnett & Wilson 1999, 2002; O’Connor et al. 2002). In the present study, where hyphal-mediated transfer was greatest between conspecific grasses, the potential for resource transfer via mycorrhizal hyphae may be a mechanism whereby this dominant, highly mycotrophic grass gains extra advantage over the less mycotrophic subdominant forb species, by minimizing intraspecific competition, improving intraspecific facilitation and maximizing intraspecific resource distribution.
However, when grown with an interspecific neighbour, both S. nutans and A. ludoviciana receiver plants took up significantly less total 32P transfer (roots and hyphae) when S. nutans was the donor species, indicating that P was limiting and that the two receiver species were competing for this resource. Although the inclusion of a neighbour resulted in less 32P transferred for both receiver species, A. ludoviciana uptake was reduced by 50%, whereas a competing neighbour reduced only 32P uptake of S. nutans by ≈26%. This indicates that the competitive effect of A. ludoviciana on S. nutans was less than the effect of S. nutans on A. ludoviciana. Thus the competitive interaction between these two species was asymmetrical, and S. nutans was a stronger competitor for P than A. ludoviciana. Furthermore, total 32P uptake (roots plus hyphae) was consistently greater for S. nutans receivers than A. ludoviciana receivers, indicating a greater ability of S. nutans to compete for P from an available pool or local mycelial network.
Grime et al. (1987) hypothesized that non-random, mycelial-mediated nutrient transfer was due to the export of assimilate from source (canopy dominants) to sink (understorey components) in response to differing tissue nutrient concentrations between plants. There has been some evidence for this phenomenon (Newman et al. 1992; Frey & Schuepp 1993), with the most convincing evidence from studies where donor plants were stressed by shoot removal or defoliation, or nutrient-poor receiver plants were grown in association with nutrient-rich (usually fertilized) donor plants (Martensson, Rydberg & Vestberg 1998; Simard et al. 2002). In the present study, tissue P concentration of the forb species was significantly greater than that of the grass. Therefore the movement of P would not be expected to move from dominant to subordinate species, as the subordinate is not a sink for P.
Although we are far from understanding the details of the links that mycorrhizal mycelial networks make in plant communities, it is plausible that most plants do enter these CMN. The results of this study indicate the potential for movement of plant resources through mycorrhizal hyphae to be an important component of the nutrient-acquisition strategy of prairie plants, and a potentially important mechanism influencing plant species interactions and community structure in grasslands, and possibly other communities. Transferring nutrients via mycorrhizal hyphae was not random, but rather favoured the dominant tallgrass prairie species, indicating that AM-mediated nutrient transfer may be a mechanism whereby this dominant, highly mycotrophic grass gains additional advantage over the less mycotrophic subdominant forb species. However, these plant species appear to have adapted to utilize different nutrient-acquisition strategies, with the facultative mycotroph relying on greater root absorption and the obligate mycotroph absorbing and transferring nutrient via mycorrhizal hyphae.