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Keywords:

  • arbuscular mycorrhizae;
  • Artemisia ludoviciana;
  • common mycorrhizal networks;
  • nutrient transfer;
  • nutrient uptake;
  • 32P;
  • Sorghastrum nutans;
  • tallgrass prairie

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • 1
    A glasshouse 32P-labelling study examined arbuscular mycorrhizal (AM)-mediated transfer of phosphorus between individuals of two tallgrass prairie species, an obligately mycotrophic grass (Sorghastrum nutans Vitm.) and a facultatively mycotrophic forb (Artemisia ludoviciana Nutt.).
  • 2
    Regardless of which species served as donor, 32P was transferred between both intra- and interspecific neighbours via AM mycelia. However, nutrient transfer via AM fungi was not uniform between neighbouring species.
  • 3
    Conservative estimates indicate that interplant transfer via AM fungi accounted for >50% of the total 32P acquisition by S. nutans, but accounted for only 20% of 32P uptake into A. ludoviciana.
  • 4
    While this study did not show conclusively that a common mycelial network acted as a conduit for nutrient transfer, it clearly demonstrated that mycorrhizae facilitated transfer.
  • 5
    The results indicate that differential movement of plant resources via AM mycelium may be a mechanism whereby a dominant, highly mycotrophic grass extends competitive advantage over a less mycotrophic, subdominant forb species in grasslands.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

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.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

soil and container preparation

A 32P tracer study was utilized to assess interplant transfer of P between a warm-season grass (Indian Grass, Sorghastrum nutans Vitm.) and a perennial forb (Louisiana Sagewort, Artemisia ludoviciana Nutt.). These two tallgrass prairie species were planted as seedlings into divided containers (120 total containers, 30 cm long × 20 cm wide × 15 cm high), constructed of plexiglass. Each container (pot) was divided into two compartments separated by either a solid plastic barrier or a 43-µm nylon mesh barrier (Tetko, Inc., Lancaster, NY, USA), or the containers were not divided. One side of each container was designated the donor side (plant labelled with 32P), the other the receiver side (32P transferred to plant from donor side). The mesh screen allowed growth of fungal hyphae from one side to the other while excluding passage by plant roots. Therefore 32P tracer acquisition by the receiver plant was attributed to hyphal-mediated uptake rather than direct uptake by plant roots. The solid plastic barriers ensured tracer labelling did not pass between compartments, and the receiver plants in these containers served as controls on which background levels of 32P could be established. For each container, 1000 g non-sterile soil was added to each compartment, with the non-separated containers receiving 2000 g non-sterile soil distributed evenly in the pot. The soil was freshly collected from tallgrass prairie areas at Konza Prairie Biological Station, Manhattan, KS, USA and transported to the glasshouses at Kansas State University. The soil was a Chase silty clay loam, fine montmorillonitic, mesic Aquic Argiudolls with a pH of 6·8. The soil contained 7 mg kg−1 plant-available P (Bray test I); 170 mg kg−1 K; 15 mg kg−1 NO3; 13 mg kg−1 NH4; and 4·2% organic matter as determined by the Kansas State Soil Testing Laboratory (Manhattan, KS, USA). Because non-sterile soil contains hyphal and spore inoculum of a variety of AM fungi, host plants may or may not have become colonized by common AM species. However, using freshly collected native soil represents both field conditions and hetero- and conspecific transfer of nutrients more closely.

mycorrhizal inoculum assessment

To assess the AM fungi present in native soil, spores were isolated from 100 g soil by wet-sieving, decanting and centrifuging in a 20 : 40 : 60% sucrose density gradient. Based on a single assessment of a 100-g subsample of soil and using the taxonomic criteria of Schenck & Perez (1990), the soil contained the following number of spores per 100 g (DM) soil: 283 Glomus heterosporum Smith & Schenck’ 266 Glomus claroideum Schenck & Smith; 167 Glomus aggregatum Schenck & Smith emend. (Gerdemann & Trappe); 128 Glomus etunicatum Becker & Gerdemann; 98 Glomus macrocarpum Tulasne & Tulasne; 86 Glomus mortonii Bentivenga & Hetrick; 61 Glomus sp. 1; 43 Glomus constrictum Trappe; 32 Entrophospora infrequens (Hall) Ames & Schneider; 31 Glomus sp. 2; 22 Acaulospora longula Spain & Schenck; 12 Glomus mosseae (Nicol. & Gerd.) Gerd. & Trappe; and 10 Scutellospora calospora (Nicol. & Gerd.) Walker & Sanders.

plant preparation

Seeds were germinated in vermiculite in a 23 °C glasshouse. One 6-week-old forb seedling (A. ludoviciana) was transplanted into the donor side of 60 containers. One 3-week-old grass seedling (S. nutans) was transplanted into the donor side of the remaining half of the containers (60). Seedlings of S. nutans or A. ludoviciana were then planted, individually or in combination with each other, into the receiver side of each pot. Therefore the receiver side of the pot contained either A. ludoviciana grown individually; S. nutans grown individually; or both A. ludoviciana and S. nutans grown together. All seedlings were transplanted on the same date. These species were selected for this study because S. nutans is a predominant matrix-forming grass and A. ludoviciana is a common co-occurring forb in tallgrass prairie (Towne 2002). Additionally, these species differ widely in their responsiveness to mycorrhizal colonization, S. nutans being classified as an obligate mycotroph (requiring the symbiosis to complete its life cycle) and A. ludoviciana a facultative mycotroph (benefiting from the symbiosis, but able to grow in its absence) (Table 1; Wilson & Hartnett 1998). The pots were arranged in a split-plot design with four replications per treatment. Plants were watered as needed and fertilized every 14 days with a dilute solution of Peter's No-Phos Special Fertilizer solution (25 : 0 : 25, N : P : K, Robert B. Peters Co., Allentown, PA, USA) providing ≈35 mg kg−1 N and ≈35 mg kg−1 K to each container.

Table 1. Sorghastrum nutans and Artemisia ludoviciana root, shoot and total plant dry weights; phosphorus concentration (mg per g plant, root + shoot) of donor and receiver plants following 14 weeks’ growth; and Bq radiation (per g plant, root + shoot) of donor plants harvested 14 days after isotope labelling (n = 360, mean ± SE)
Parameter*Plant species
A. ludovicianaS. nutans
  • *

    Significant difference between A. ludovicina and S. nutans as determined by paired t-tests (P ≤ 0·05). 32P values were log-transformed prior to analysis, actual values are presented.

  • **

    Mycorrhizal responsiveness (%) = [(mean DM mycorrhizal plant − mean DM non-mycorrhizal plant)/mean DM mycorrhizal plant] × 100. Values are from Wilson & Hartnett (1998).

Mycorrhizal responsiveness (%)**44·399·5
Root DW (g)0·82 (±0·02) 0·87 (±0·02)
Shoot DW (g)3·69 (±0·05) 3·78 (±0·05)
Total DW (g)4·52 (±0·06) 4·65 (±0·05)
P concentration (µg per g plant) 945 (±174) 585 (±131)*
32P uptake of donor plant (Bq per g plant) 849 (±185)1343 (±154)*

fungicide applications

Seedlings were allowed to establish and become colonized by indigenous mycorrhizal fungi (contained in the non-sterile prairie soil) for 9 weeks. To assess P transfer from the donor to the receiver plant, attributable solely to root uptake (leaked from donor roots and subsequently taken up by receiver roots), the symbiosis was suppressed in divided and non-divided containers. The suppression treatment was implemented by adding 500 ml of a 50 µg g−1 (active ingredient) benomyl solution (Benlate) to the soil of each container after 9 and 11 weeks’ plant growth. An equal amount of water (500 ml per container) was applied to the controls. The fungicide benomyl has been used successfully in our studies and by others to reduce mycorrhizal activity in field and glasshouse experiments (Merryweather & Fitter 1996; Hartnett & Wilson 2002; O’Connor, Smith & Smith 2002), as well as nutrient transfer studies (Jakobsen, Grazey & Abbott 2001; Schweiger & Jakobsen 2001). Evidence from a wide array of studies indicates that, in tallgrass prairie ecosystems, the use of benomyl is a conservative approach, its primary effect being suppression of mutualistic mycorrhizal associations and their symbiotic function (Wilson & Hartnett 1997; Hartnett & Wilson 1999; Hartnett & Wilson 2002; O’Connor et al. 2002).

isotope labelling

Eleven weeks after transplanting seedlings into containers, a foliar feeding technique was used to label donor plants with 32P without contaminating the surrounding soil. Plants were labelled with 32P as KH2PO4. The ends of two healthy, mature leaves of each donor plant were cut at an angle and immersed in 20 ml aqueous solution containing 3·0 × 106 Bq 32P. The feeding tubes were plugged with cotton to secure leaves within the tubes, sealed to prevent spillage, and clamped to wooden rods. After 14 days the immersed leaves were severed 5 cm above the top of the sealed tubes, and the tubes were removed. Plants were harvested and the roots washed free of soil. Shoots and roots were oven-dried at 60 °C for 72 h for root, shoot and total dry weight determination. To determine mycorrhizal colonization, subsamples of dried roots were stained in trypan blue using the method of Koske & Gemma (1989), and scored for AM colonization using the magnified gridline intersect method developed by McGonigle et al. (1990). Root subsamples were then destained in 0·5% sodium hypochlorite, rinsed in distilled water, and dried at 60 °C for 24 h. Dried root and shoot samples were ground, passed through a 2-mm-mesh sieve, and digested in double acid (HNO3–HClO4 at a 1 : 1 v : v ratio). Digested samples were analysed for P concentration (phosphomolybdate method with ascorbic acid reduction). Digested samples were mixed with 5 ml scintillation cocktail (1 : 1 v : v for 32P quantification using a scintillation counter). Counts were corrected for background and decay. Background levels of 32P were established using the mean radioactivity level from receiver plants of the plastic barrier treatment.

statistical analyses

Shoot, root and total plant DW; AM root colonization; and root, shoot and total plant P and 32P concentrations and uptake (concentration × DW) data were tested for homogeneity of variance prior to analysis. Counts of radiation were log10-transformed (x + 1) prior to analysis to reduce heterogeneity of variances. All other variances were determined to be homogeneous according to Levine's test for homogeneity of variance. Data were analysed as a split-plot design using proc mixed (SAS Institute, Inc.), with fungicide/barrier combinations as whole-plot treatments and donor/receiver species combinations as subplot treatments. Significant effect means (P ≤ 0·05) were separated by a paired t-test. Because total plant dry weights were not significantly different, regardless of treatment or plant species, plant tissue 32P concentration (Bq per g plant biomass) and total plant 32P uptake (concentration × DW) measurements were highly correlated (P < 0·0001, r = 0·799), thus only 32P-uptake data are presented.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

dry weight and total tissue p

Within plant species there were no significant effects of fungicide application on either plant biomass production or plant tissue P, presumably because fungicide application occurred only during the final 2 weeks of the experiment, and because it has no direct effect on plant growth. Analysis of variance for root, shoot or total plant biomass production of S. nutans and A. ludoviciana revealed no significant effects of root barrier treatments, nor were there differences in dry weight between plant species (Table 1). Plant tissue P concentration was greater for A. ludoviciana than S. nutans (F1,63 = 145·7, P < 0·0001; Table 1).

Root colonization by mycorrhizal fungi

Mycorrhizal root colonization displayed a treatment × donor plant species interaction (F2,63 = 28·0, P < 0·0001; Table 2). For untreated soils (not receiving fungicide), significantly higher levels of colonization of roots were observed for S. nutans than A. ludoviciana (36·0 vs 20·9%, respectively, of the total root length colonized; t63 = 8·8, P < 0·0001). The application of benomyl to S. nutans plants significantly reduced root colonization from 36·0% of total root length in the non-fungicide controls to only 6·7% in the fungicide-treated pots, an 81% reduction in colonization relative to mycorrhizal controls (t63 = 17·9, P < 0·0001). Similarly, the application of fungicide reduced the percentage root colonization of A. ludoviciana from 22·0% in the control pots to 8·2% in the fungicide-treated pots, a 61% reduction in colonization relative to controls (t63 = 10·1, P < 0·0001).

Table 2. anova for effects of barrier or fungicide on mycorrhizal colonization, phosphorus concentration and 32P uptake of Sorghastrum nutans and Artemisia ludoviciana receiver plants
EffectF
Colonization (%)P concentration (µg per g plant)32P uptake of receiver plant (Bq per g plant)
  • ***

    , P ≤ 0·001;

  • *

    , P≤ 0·01.

  • Whole-plot treatment = barrier or fungicide or non-amended control.

Whole-plot treatment (T)256·42***  0·16138·17***
Donor species (D)145·71***  0·09530·46***
Receiver species (R)  1·55145·72***  2·01
D × R  1·42  0·00 25·11***
Neighbour within receiver [N(R)]  0·11  1·03  2·51
D × N(R)  0·08  0·53  3·52*
T × D 27·97***  0·12  0·73
T × R  0·56  0·46 98·76***
T × D × R  0·16  0·09  0·07
T × N(R)  0·59  0·19  2·79
T × D × N(R)  0·94  0·13  1·63

32p labelling and uptake

The shoots of donor plants retained high counts of radiation (1343 and 849 Bq for S. nutans and A. ludoviciana, respectively; Table 1), indicating that our foliar-feeding technique was successful in labelling donor plants with 32P. When donor and receiver plants were separated by a plastic (impenetrable) divider, low levels of radiation were detected in roots or shoots of receiver plants at harvest (<0·1 Bq in root tissue; <0·15 Bq in shoots), with no variation due to species. Therefore background levels of 32P were established using mean values for root, shoot and total counts of radiation of receiver plants from this treatment.

In plants separated by a 43-µm-mesh barrier, uptake of 32P into receiver plants following fungicide application was not significantly different from background radiation levels (plastic barrier) (t63 = 0·07, P = 0·97). Therefore no transfer of 32P isotope occurred via diffusion or mass flow through the soil, and the radiation transferred to receiver plants in non-fungicide-treated pots was hyphal-mediated. The lack of transfer when fungicide was applied also confirms that, in the treatment with unseparated donor and receiver plants, transfer was primarily due to root uptake following fungicide treatments (isotope leaked into the soil from donor roots taken up by roots of the receiver). This allows us to use the treatment combinations (Table 3) to assess the relative contribution of root vs hyphae to the uptake of 32P isotope from donor to receiver plants and direct hyphal transfer. When donor and receiver plants were not separated by either barrier, and no fungicide was applied, the uptake into receiver tissues is attributable to the combination of root uptake of isotope leaked from donor roots or hyphae into the soil; hyphal uptake of leaked isotope; and interhyphal transfer.

Table 3.  Experimental design for examination of phosphorus transfer from donor plant to receiver plant and proposed mechanism of nutrient transfer
Barrier separating donor and receiver plantsSoil treatmentProposed P-transfer mechanism
Plastic barrierNo treatmentNo transfer
No barrierNo treatmentRoots and hyphae
No barrierFungicideRoots
43-µm barrierFungicideNo uptake
43-µm barrierNo treatmentHyphae

32P uptake of receiver plants displayed interactions between donor species and receiver species combinations (F2,56 = 3·52, P = 0·036), and between treatments and receiver species combinations (F2,56 = 2·8, P = 0·035; Table 2). Regardless of donor species, A. ludoviciana accumulated more 32P than S. nutans when acquisition was via root only (fungicide-treated with no barrier), whether grown individually or in combination (Fig. 1a,b). In contrast, in the treatments where rooting-zone overlap was prevented but mycorrhizal network connections were maintained (43-µm-mesh barrier but no fungicide), uptake was greatest (P ≤ 0·05) in S. nutans receiver plants, whether S. nutans or A. ludoviciana was the donor species. When uptake was attributable to roots and hyphae (no fungicide and no barrier), isotope abundance of the receiver was greatest in S. nutans when S. nutans was also the donor species, but only when not grown in combination with A. ludoviciana (Fig. 1a). When either S. nutans or A. ludoviciana was the receiver, total 32P uptake was reduced (P ≤ 0·05) when paired with a neighbour (Fig. 1a). Under the similar set of treatments, but with A. ludoviciana as the donor species (Fig. 1b), this similar general pattern of root vs hyphal uptake was evident, but the differences were much smaller.

image

Figure 1. Total 32P uptake for Sorghastrum nutans and Artemisia ludoviciana receiver plants grown together or separately. The donor species was S. nutans (a) or A. ludoviciana (b). Total =32P transfer into receiver plant when donor and receiver roots were grown together; roots = 32P transfer into receiver plant when donor and receiver roots were grown together and mycorrhizal activity was suppressed by fungicide applications; mycorrhizae = mycorrhizal-mediated 32P transfer into receiver plant when donor and receiver roots were separated by a 43-µm-mesh root barrier. Receiver plants: S. nutans grown alone (solid bars); S. nutans grown with A. ludoviciana (hatched bars); A. ludoviciana grown alone (open bars); A. ludoviciana grown with S. nutans (stippled bars). Different letters above bars indicate significant difference (P ≤ 0·05); error bars, ±1 SE.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

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.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This paper is contribution Number 06-43-J from the Kansas Agricultural Experiment Station, Kansas State University, Manhattan, Kansas, USA. The research was partially supported by the National Science Foundation (Grant DEB-9873654) and the National Science Foundation Long-Term Ecological Research Program (Grant IBN-9632851). We would like to thank Timothy C. Todd, Kansas State University Department of Plant Pathology, for statistical advice and R. Michael Miller, Argonne National Laboratories, for comments and discussion on this manuscript.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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