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Keywords:

  • biodiversity hotspot;
  • cluster roots;
  • manganese;
  • microcosm;
  • mycorrhizal networks;
  • nutrient-poor soils;
  • phosphorus;
  • Proteaceae;
  • root competition

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Concluding remarks
  8. Acknowledgements
  9. References
  10. Supporting Information
  1. Greater understanding of positive interspecific interactions in nutrient-poor soils is a priority, particularly in phosphorus (P)-limited ecosystems where plants with contrasting nutrient-acquiring strategies co-occur. It is also relevant to agro-ecosystems, since global P stocks are being depleted. In this study, we assess positive interactions between sympatric plants with contrasting nutrient-acquiring strategies from highly P-impoverished soils from the biodiversity hotspot of south-western Australia.
  2. Four plant species (Banksia menziesii, Eucalyptus marginata, Verticordia nitens and Melaleuca preissiana) that are non-mycorrhizal (cluster-rooted), ectomycorrhizal (EM), arbuscular (AM) or dual AM/EM, respectively, were grown together in a specially designed ‘common garden’ microcosm with nutrient-poor or fertilized soil, with or without root intermingling and fungal hyphae contact. We measured growth, mycorrhizal colonization, root intermingling and nutrient uptake to determine positive or negative growth patterns amongst the various plant assemblies.
  3. Growth of the AM/EM host was best when interacting with both the EM host and a non-mycorrhizal nutrient-mining plant with cluster roots (Banksia) in microcosms where root intermingling was not possible. Growth promotion was only seen in pots with nutrient-poor soils, where the better growth of Melaleuca coincided with higher shoot P, manganese, calcium, iron and boron content, whereas an increase in soil nutrient status through fertilizer addition resulted in a decrease in nutrient-sharing between co-occurring species. Furthermore, the dual AM/EM Melaleuca exhibited enhanced EM colonization and favoured EM over AM fungi when grown beside Eucalyptus and Banksia. We surmise that mycorrhizal networks were instrumental in the variation in both mycorrhizal type and colonization levels.
  4. We conclude that complementarity of plant nutrient-acquisition strategies can promote growth of neighbour species. The results show a synergistic effect between EM hyphal scavenging and mobilization of limiting nutrients by cluster roots. The positive and negative interactions enable coexistence to go far beyond the traditional view that plants interact mainly through resource depletion. This study improves our understanding of how root interactions could shape plant communities and promote species diversity and packing in nutrient-impoverished habitats.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Concluding remarks
  8. Acknowledgements
  9. References
  10. Supporting Information

Plant communities may express both competition and facilitation, where at least one species provides conditions that inhibit or favour the presence and growth of another, respectively (Callaway 2007; Butterfield & Callaway In Press). Plant root interactions may be intensified between plants that require the same limiting resources or where a change in the availability of those resources is altered by soil biota (e.g. mycorrhizal fungi). It is unclear, however, whether functional diversity in terms of nutrient-acquiring strategies, including different mycorrhizal and non-mycorrhizal strategies, may contribute to enhanced plant–plant interactions.

Plants can enhance the growth of other nearby plants by ameliorating harsh environmental conditions (Bertness & Callaway 1994; Callaway 2007) or by increasing the availability and/or uptake of nutrients (Lambers, Chapin & Pons 2008a; Muler et al. 2014). Indirect positive interactions between plants can occur, for example, when one plant fixes nitrogen (N2) symbiotically and enriches the surrounding soil for nearby interacting plants (van der Putten 2009). Phosphate is generally poorly mobile in soil and effective acquisition of P by plants generally does not simply depend on mass flow and diffusion, but requires additional strategies, for example mycorrhizas (Marschner 2012). The release of P from organic compounds via root exudation of enzymes (Li et al. 2003) or the dissolution of P in soil due to exudation of carboxylates (Gardner, Barber & Parbery 1983) are other examples of enhanced nutrient uptake. The acidification of the rhizosphere due to carboxylate exudation also increases micronutrient availability, particularly manganese (Mn) (Muler et al. 2014). In agricultural plants, positive interacting processes are known (Zuo et al. 2000; Li et al. 2007); however, there are important gaps in our understanding of the role of these interactions for species coexistence in native plant communities in nutrient-limited ecosystems.

Mycorrhizal interactions in plants may boost plant fitness leading to an increase in reproductive output (seed) and seed quality (Francis & Read 1994; Smith & Read 2008). Ectomycorrhizal networks can promote the colonization of neighbouring plants interacting with a well-colonized focal plant (Simard et al. 2012). Ectomycorrhizal networks that form between plants sharing the same fungal type may also enhance transfer of nutrients between plants (Teste et al. 2009; Simard et al. 2012), by direct anastomosis (Giovannetti et al. 2004), hyphal leakage and hyphal turnover (Staddon et al. 2003), and are thus a below-ground form of growth-promoting interaction. Positive effects through mycorrhizal networks are expected when nutrient sinks develop in one of the interacting plants, thus indirectly promoting subsequent nutrient uptake (van der Heijden & Horton 2009; Simard et al. 2012). Such below-ground positive interactions would be expected to be particularly of ecological relevance in nutrient-poor soils, where nutrients such as P are bound in pools resident in plant tissue and micro-organisms (Turner et al. 2013), but this has never been investigated.

Traditionally roots have been thought to interact in nutrient-rich patches or avoid interacting by exploring unoccupied space (de Kroon, Mommer & Nishiwaki 2003). In nutrient-poor soils, where nutrients may be homogeneously distributed (i.e. without many nutrient-rich patches), roots are expected to explore more soil volume without local proliferation and potentially exhibit spatial segregation with depth (Hodge 2004). However, roots also respond to the presence of neighbouring roots by competing (Mommer et al. 2012) or perhaps by proliferating around neighbouring ‘leaky’ (i.e. exuding nutrients into the rhizosphere) roots (Fustec et al. 2010). Furthermore, some plants may allow their mycorrhizal fungal hyphae to interact with neighbouring ‘leaky’ roots, although this mechanism is not well documented and requires empirical support; such a system is established for some mycoheterotrophic orchids (Dixon, Pate & Kuo 1990). It is unclear how these driving factors for plant nutrient uptake affect interactions amongst plants, and ultimately plant growth and plant diversity in nutrient-poor soils where plants with different nutrient-acquisition strategies coexist (Lambers et al. 2013).

The objective of this study was to investigate root interactions and nutrient uptake amongst sympatric plant species with four distinct nutrient-acquisition strategies; arbuscular mycorrhiza (AM), ectomycorrhiza (EM), dual AM/EM and non-mycorrhizal cluster roots (Lambers et al. In press). First, we hypothesized that a nutrient-mining plant with cluster roots would promote growth of one or more of the sympatric plant partners by enhanced nutrient acquisition, in particular P and Mn; secondly, that the mycorrhizal type (e.g. EM or AM) of a plant would influence the mycorrhizal type of neighbouring plants capable of forming dual AM/EM associations; and finally, that promotion of growth would be more pronounced in nutrient-poor than in fertilized soils.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Concluding remarks
  8. Acknowledgements
  9. References
  10. Supporting Information

Experimental setup

Plants of four native sympatric species from the biodiversity hotspot of south-western Australia were grown together in groups of three plants of different species in microcosm pots. The seedlings [Melaleuca preissiana Schauer (Myrtaceae), Eucalyptus marginata Sm. (Myrtaceae), Verticordia nitens (Lindl.) Endl. (Myrtaceae) and Banksia menziesii R.Br. (Proteaceae)] were grown from seed in commercial production nurseries for 8 months. These four species are referred to hereafter by their generic names. Melaleuca was selected since we expected it to form dual AM/EM strategies, while Eucalyptus and Verticordia have been reported to be EM and AM, respectively (Brundrett 2009). Finally, Banksia was chosen since it is a non-mycorrhizal plant using cluster roots as a strategy for nutrient acquisition (Lambers, Chapin & Pons 2008a). We also chose these four plant species because they are widespread and common, and also representative of widely occurring, contrasting nutrient-acquisition strategies (Lambers et al. 2006). They are also long-lived woody perennials that have similar growth rates as early-stage seedlings (F.P. Teste, pers. obs.) and do not differ in any other key confounding traits (Pate & Bell 1999), thereby minimizing potential idiosyncratic results. Similar-size seedlings were then transplanted into large microcosm pots containing a soil mix (see ‘Growth substrate’ below) and grown for 7 months with or without fertilization. The microcosm pots consisted of three fused PVC cylinders, one wider than the other two with the planes of fusion fully open or with a 50-μm mesh that prevented root intermingling between species (Fig. 1). Forty-eight Melaleuca grew in the main compartment and acted as focal plants since Melaleuca could form dual AM/EM strategies. The two smaller compartments grew combinations of the three other plant species; either Banksia with Verticordia, Eucalyptus with Banksia or Eucalyptus with Verticordia.

image

Figure 1. Drawing of one of the microcosm pots where a main compartment and two smaller compartments were assembled to grow three interacting plants of different species with or without mesh barriers. Shown here is a microcosm pot with Melaleuca preissiana (focal plant) in the main compartment, Banksia menziesii in the left smaller compartment and Verticordia nitens on the right. Total microcosm volume: 11 680 cm3, where the main compartment volume was 6180 cm3 and the two ‘piggy-back’ smaller compartments were 2750 cm3 each. In the pots without mesh, the window size that allowed roots to intermingle was 5 cm wide by 34 cm long.

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Growth substrate

Soils were collected at ten random locations proximal to AM and EM plants at two bushland sites with the aim to maximize mycorrhizal inoculum potential while maintaining a growing substrate representative of the soils found in the distributional range of the plant species (Table 1). Spearwood sand was collected from native woodland at The University of Western Australia Shenton Park Field Station (31·96°S, 115·79°E) near Perth. The field station hosts a woodland dominated by the trees, Banksia attenuata, B. menziesii, Eucalyptus marginata and shrubs/arborescent monocots Allocasuarina fraseriana, Xanthorrhoea preissii and Hibbertia hypericoides. Jarrah forest soil was collected near Alcoa's Huntly mine (32·33°S, 116·07°E) in undisturbed forest dominated by E. marginata. We sieved and homogenized equal parts of Spearwood sand, Jarrah soil, washed river sand (see Pate & Bell (1999) for properties), and Perlite to be used as the growing substrate. The soil mix was immediately used to maximize mycorrhizal inoculum in the soil. Finally, fertilizer (using the proprietary Osmocote® slow-release fertilizer specifically designed for Australian native plants N:17·9, P:0·8, K:7·3; added 23·4 g NPK per pot with trace elements) was added to half of the microcosm pots.

Table 1. Soil nutrient concentrations and chemical characteristics at the start of the experiment. These data show that plants were grown in nutrient-impoverished soils typical of the Swan Coastal Plain soils of south-western Australia based on representative soil nutrient levels previously measured (McArthur, Johnston & Snell 1991; Laliberté et al. 2012)
MacronutrientsNPKMgCaS
Concentrationa
Mean0·46228·780·72838
Standard deviation (SD)0·00302·19·1321
MicronutrientsMoCuZnFeMnBNi
Mean00·130·911318·70·130·067
SD00·060·162·10·060·058
Other elements and characteristicsAlCoNaECpH
  1. a

    All soil nutrient concentrations are in mg kg−1 except for N, whose concentration is in g kg−1. Electrical conductivity (EC) is in mS m−1; pH was measured in CaCl2.

Mean5270·1124·33·674·53
SD310·012·30·580·06

Soil analysis

At the start of the experiment, three air-dried and sieved (2 mm) soil samples from the soil mix were sent to the ChemCentre (Perth, Australia) for analysis of nutrients, electrical conductivity and pH. The soil was subjected to the Mehlich 3 extraction (2·5 g + 25 mL) for 5 min (Rayment & Lyons 2010) and then analysed for extractable P, K, Ca, Mg, Na, S, B, Co, Cu, Fe, Mn, Mo, Ni and Zn by ICP-AES (Varian Vista axial spectrometer, Palo Alto, CA, USA). Total N was quantified with a Technicon AA Auto Analyser and Multielement Varian Axial Optical Emission Spectrometer after Kjeldahl acid-digestion (Blakemore 1981). Soil pH was measured using a glass electrode in a 1 : 5 extract of soil and 0·01 m CaCl2 (Blakemore 1981). Electrical conductivity (EC) was measured with an EC metre in DI water.

Seedling measurements and foliar analysis

At the start and end of the experiment, seedlings were destructively harvested to measure height, stem diameter at the base, shoot and root dry biomass, mycorrhizal colonization, foliar nutrient concentrations and root intermingling. Harvested shoots were dried at 70 °C for 48 h, ground and analysed for N by the Kjeldahl digest method (Blakemore 1981) and P, K, Na, Ca, Mg, S, Al, B, Cu, Fe, Mn, Mo and Zn, after combustion by atomic absorption spectrometry (Perkin Elmer 500 DV ICP-AES), at the ChemCentre, Perth, WA, Australia.

Root intermingling

The roots from the microcosms without the mesh barrier were carefully harvested using a novel ‘sever and fishing’ approach to account for root intermingling. In brief, a sharp and flexible handsaw was constructed to cleanly sever the roots crossing over to neighbouring compartments. Whole root systems were then carefully extracted and washed with tap water over a fine sieve. Severed loose root fragments were collected in the sieve and were assumed to be roots of the plant in the neighbouring compartment. Finally, the intermingling root fragments were placed into an 80% (v/v) ethanol solution for scoring into species under a dissecting microscope.

Root staining and mycorrhizal colonization

One subsample of lateral roots was randomly sampled from all individual plants. Roots were cleared and stained using the ink and vinegar staining technique for mycorrhizal fungi (Vierheilig et al. 1998) with the following modifications. Roots were carefully washed with deionized (DI) water, cut into 1–2 cm fragments, homogenized and placed in a 10% (w/v) KOH solution for 5 h at 90 °C in at least three times more KOH solution than root volume in glass tubes. Then, cleared roots were rinsed twice with DI water, bleached (1 : NH4OH, 1 : H2O2, 200 : DI H2O) at room temperature for 5–45 min, depending on root ‘whiting’. Bleached roots were then rinsed twice with DI water and placed in an ink vinegar solution (5 : black ink, 100 : 5% (v/v) acetic acid) at 100 °C for 5 min for staining. Stained roots were then rinsed three times in DI water and transferred to acidified water (pH c. 3) for 30 min. Finally, acidified roots were placed in 50% (v/v) lactoglycerol (1 : lactic acid, 1 : glycerol, 1 : H2O) solution for destaining, scoring and storage.

Arbuscular mycorrhiza fungal colonization was determined in cleared and stained roots using the gridline intersect method (Giovannetti & Mosse 1980). Ectomycorrhizal fungal colonization was determined based on counts of all root tips present in the counting dish. Therefore, all root tips (non-EM and EM tips) were counted until 100 EM or 100 non-EM was reached, thus giving values to calculate percentage colonization following Teste et al. (2006). Briefly, percentage EM colonization = the number of active EM root tips/(the number of active EM root tips + the number of active non-EM root tips) multiplied by 100.

Data analysis

Statistical analyses utilized the r statistical computing and graphics programme (R Core Team 2013). The data were analysed as two experiments: one for plants grown in unfertilized soils and the other for fertilized soils. A fixed-effects model was used with a factorial treatment structure where the factors mesh (two levels: with or without), and neighbouring plants (Eucalyptus & Verticordia, Eucalyptus & Banksia, Verticordia & Banksia) with four replicate microcosms (n = 4). Equal variance and normality were assessed graphically in r. The pooled variance and experimental error degrees of freedom were used to calculate Tukey's honestly significant difference (Tukey HSD) error bars and 95% confidence intervals (CIs) for statistical and ecological inferences (Altman et al. 2000; di Stefano, Fidler & Cumming 2005). In figures, the CIs were accompanied by Tukey HSD bars (α = 0·05) and used as visual multiple-mean comparison tests to detect statistically significant effects and highlight precision and uncertainty in the estimates (Altman et al. 2000). Results of null hypothesis significance testing and P values were generally not reported, because Tukey HSD error bars and CIs provided sufficient information needed for conducting inferences, determining uncertainty and suggesting ecological importance of the effects (Cumming 2008).

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Concluding remarks
  8. Acknowledgements
  9. References
  10. Supporting Information

In nutrient-poor soils, Melaleuca shoots were most vigorous when grown beside E. marignata and B. menziesii separated by the root-excluding mesh barrier (Figs 2a & S1, Supporting information). The greatest net biomass gains in roots of Melaleuca were also found when growing beside Eucalyptus and Banksia, compared with growth beside Eucalyptus and V. nitens only; the Tukey HSD bars overlapped in the other comparison, regardless of the mesh treatment (Fig. 2b). Similar to the net root biomass results, height increment of Melaleuca growing beside Eucalyptus and Banksia tended to be greater than that in the other plant assemblies (Fig. S1). In soil with fertilizer, as expected, net biomass gains of plants were considerably greater; however, positive or negative plant–plant interactions were no longer detected (Table S1–S2, Supporting information).

image

Figure 2. Net biomass gain (a: shoots, b: roots) of Melaleuca preissiana (Mp) as the focal plant in a microcosm with combinations of Em, Eucalyptus marginata; Vn, Verticordia nitens; Bm, Banksia menziesii. For the root biomass, there was no-mesh treatment effect. The mean biomass of five randomly selected focal plants at the start of the experiment was used as initial biomass value. Values are means with 95% confidence intervals (dotted lines) with Tukey's HSD error bars (solid). Statistically different means can be assessed visually; they exist when two Tukey's HSD error bars do not overlap.

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Shoot P and Mn content of Melaleuca were greatest when Melaleuca grew beside Eucalyptus and Banksia, matching the growth responses and suggesting that the increased nutrient uptake promoted by neighbouring Eucalyptus and/or Banksia (Fig. 3) was at least partly responsible for the enhanced growth of this plant assembly. The greater shoot P and Mn content did not always result in higher concentrations of these elements: only when growing beside Eucalyptus and Banksia compared with Banksia and Verticordia without mesh did Melaleuca show the greatest concentration of Mn in shoots (Fig. 4). In pots with mesh, Melaleuca with Eucalyptus and Banksia had lower concentrations of P only compared with the assembly with Eucalyptus and Verticordia (hosts of EMF and AMF) (Fig. 4). Other macro- and micronutrients such as Ca, Fe and B showed similar results, indicating that enhanced nutrient uptake occurred for both limiting and non-limiting elements (Fig. S2, Supporting information).

image

Figure 3. Shoot phosphorus (P) (a) and manganese (Mn) (b) content of Melaleuca preissiana (Mp) as the focal plant in a microcosmos with combinations of Em: Eucalyptus marginata, Vn: Verticordia nitens and Bm: Banksia menziesii. Values are means with 95% confidence intervals (dotted lines) with Tukey's HSD error bars (solid). Statistically different means can be assessed visually; they exist when two Tukey's HSD error bars do not overlap.

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image

Figure 4. Shoot phosphorus (P) (a) and manganese (Mn) (b) concentration of Melaleuca preissiana (Mp) as the focal plant grown with combinations of Em: Eucalyptus marginata; Vn: Verticordia nitens; and Bm: Banksia menziesii. Values are means with 95% confidence intervals (dotted lines) with Tukey's HSD error bars (solid). Statistically different means can be assessed visually; they exist when two Tukey's HSD error bars do not overlap.

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The net biomass gain of Melaleuca growing beside Eucalyptus and Banksia in the pots with mesh showed a strong relationship with the root biomass of these neighbouring plants (Fig. S3, Supporting information). This was not the case in the pots without mesh where roots could freely intermingle (Fig. S3). This result indicates greater scavenging by ectomycorrhizal fungi in that assembly as suggested below and shown in Figs 5 and 6.

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Figure 5. Boxplots of arbuscular (AM) and ectomycorrhizal (EM) fungal colonization of Mp, the focal plants at the end of the experiment. Mp: Melaleuca preissiana; Em: Eucalyptus marginata; Vn: Verticordia nitens; and Bm: Banksia menziesii.

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image

Figure 6. Ectomycorrhizal fungal colonization of Melaleuca preissiana (Mp) as the focal plant at the end of the experiment. Em: Eucalyptus marginata; Vn: Verticordia nitens; and Bm: Banksia menziesii. Values are means with 95% confidence intervals (dotted lines) with Tukey's HSD error bars (solid). Statistically different means can be assessed visually; they exist when two Tukey's HSD error bars do not overlap.

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Ectomycorrhizal fungal colonization of Melaleuca growing beside Eucalyptus and Banksia was greater than AMF colonization (Figs 5 and 6). Ectomycorrhizal fungal colonization of Melaleuca was considerably greater in this assembly compared with the assembly with Verticordia and Banksia, likely due to Eucalyptus being a preferential EMF host plant (Fig. 6). With fertilization, there were no differential colonization levels of Melaleuca amongst the various plant assemblies (data not shown).

Root intermingling overall was greater when Melaleuca grew beside Eucalyptus and Banksia compared with growing beside Eucalyptus and Verticordia (Fig. 7). However, only 1·4% of Melaleuca roots were found intermingling in the neighbouring compartments. This result reinforces that the evidence for enhanced nutrient uptake was not solely due to fungal interactions, but also due, but to a lesser degree, to the amount of root intermingling in the soil. Finally, fertilization reduced the amount of root intermingling in all assemblies, suggesting plant–plant interactions are more pronounced in nutrient-poor soils (Fig. 7).

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Figure 7. Biomass of roots of Melaleuca preissiana (Mp) found growing into compartments occupied by neighbouring plants Em: Eucalyptus marginata; Vn: Verticordia nitens; and Bm: Banksia menziesii. Values are means with 95% confidence intervals (dotted lines) with Tukey's HSD error bars (solid). Statistically different means can be assessed visually; they exist when two Tukey's HSD error bars do not overlap.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Concluding remarks
  8. Acknowledgements
  9. References
  10. Supporting Information

Positive species interactions in nutrient-poor soils

We observed enhanced growth only when the focal plant Melaleuca grew beside an EMF host (E. marginata) and a nutrient-mining plant with cluster roots (B. menziesii) in a mesh microcosm where root intermingling was not possible in pots with nutrient-poor soils. The enhanced growth found for Melaleuca coincided with a higher P, Mn, Ca, Fe and B content of the shoots. These results suggest a positive fungal mediated interaction mechanism, where enhanced nutrient acquisition is likely and depended on the combination and complementarity of distinct nutrient-acquiring strategies of neighbouring plants. This kind of positive root interaction amongst plants is perhaps an under-appreciated mechanism partly responsible for plant coexistence in plant communities growing in severely nutrient-impoverished soils. Mycorrhizal networks, as an example of a fungal mediated interaction mechanism, have been demonstrated to function differently with different plant assemblies, which corroborates our findings (Walder et al. 2012). Higher P, Mn, Ca, Fe, and B content did not coincide with increased concentrations of these nutrients, most likely because the concentrations of some of these nutrients were severely limiting plant growth, thus any increased uptake would lead to increased growth only (de Groot et al. 2001, 2003).

Positive interactions are typically observed under harsh environmental conditions, and it is referred to as ‘context dependent’ (Brooker et al. 2007; Dickie et al. 2007; van der Heijden & Horton 2009). Here, for the first time, we show that probable growth promotion is also dependent on neighbouring plants with different nutrient-acquisition strategies including a non-mycorrhizal strategy. We also demonstrate that this probable growth-promoting effect is dependent on soil nutrient levels, where positive or negative plant interactions are observed only when nutrients are limiting plant growth. In crop plants, analogous improved mineral nutrition has been shown when one of the two interacting plants fixes N2 when grown in mixtures (Hauggaard-Nielsen & Jensen 2005; Gunes et al. 2007; Li et al. 2007). The possibility therefore exists that as nutrients levels in soils decline, there is an increase in the ability to ‘pack’ more species into a given area via nutrient-sharing arrangements, rather than the principle of ‘winner takes all’ that is a phenomenon of eutrophic systems (Hautier, Niklaus & Hector 2009).

A few root-mediated mechanisms may have been responsible for the enhanced nutrient uptake that we observed. There was likely greater fungal scavenging capability of Melaleuca due to higher and preferential EMF colonization brought upon by the neighbouring EMF host plant Eucalyptus. Melaleuca formed both types of mycorrhizas with either AMF or EMF or both simultaneously, but in the presence of Eucalyptus, Melaleuca was preferentially colonized by EMF, perhaps due to suppressive effects of AMF when grown in the presence of Verticordia (Janos et al. 2013). This more extensively colonized root system presumably allowed Melaleuca to more effectively scavenge the nutrients mobilized by cluster-rooted Banksia (Muler et al. 2014). However, the release of limiting nutrients into soil by neighbouring plants could also have resulted from rhizodeposition or phosphatase activity (Walker et al. 2003; Lynch & Brown 2008), but transfer of nutrients back to the focal plant Melaleuca was ultimately mediated by EMF.

The greater root biomass of Melaleuca when grown with Eucalyptus and Banksia in the nutrient-poor soils further supports a facilitative capacity of the root interaction. Specifically, the mechanism entailed the mobilization of limiting macronutrient P and limiting micronutrient Mn (Muler et al. 2014) by cluster roots of Banksia in the presence of nearby EMF-scavenging hyphae connected to Melaleuca. Similarly, a recent study using Banksia attenuata showed that cluster-root activity promoted the acquisition of nutrients by neighbouring AMF host Scholtzia involucrata (Muler et al. 2014). Our study extends our understanding of enhanced nutrient uptake to plant communities with EMF and suggests a greater positive effect of EM on growth and nutrient uptake compared with AM plants. Thus, we provide evidence to support the claims that EM plants can withstand poorer soils compared with AM plants (Lambers et al. 2008b).

Mycorrhizal networks may also have enhanced nutrient uptake, since the possible growth-promoting effects were preferentially seen when the plants were separated by a root-restricting mesh barrier. In the no-mesh treatment, positive growth effects due to mycorrhizal networks could still have operated, but their magnitudes were likely reduced by root competition. Furthermore, the root intermingling and scavenging capability of non-mycorrhizal Banksia was more prominent in the no-mesh treatment, thereby potentially reducing the occurrence of mycorrhizal networks as a result of more non-mycorrhizal cluster roots dominating the intermingling space. However, hyphal links that could form between Melaleuca and Eucalyptus may have transferred nutrients bidirectionally when grown with Banksia, the non-mycorrhizal species, as the third partner. In this case, the EMF on the Eucalyptus host plant may have scavenged nutrients in the vicinity of Banksia cluster roots, and then connected the roots of the Melaleuca focal plant via mycorrhizal hyphae or rhizomorphs. Transfer of nutrients from Eucalyptus to Melaleuca could then have been promoted by a relatively large sink for nutrients in a larger faster-growing Melaleuca focal plant (data not shown).

When grown with Eucalyptus and Banksia, Melaleuca showed greater shoot P, Mn, Ca, Fe and B contents, possibly explaining the positive growth effect. Enhanced uptake of key limiting nutrients such as P and Mn (Gardner, Barber & Parbery 1983; Gardner & Boundy 1983) may not be the only mechanism explaining the enhanced growth, since shoot concentrations were less consistent than the shoot content data. However, dilution of acquired nutrients certainly occurred, since nutrient levels were only measured on whole shoots that included woody stems, which lowered the concentration values. Furthermore, lack of coincident P and Mn concentrations does not dismiss the evidence against ‘promoted uptake’ since plant biomass gains are not invariably correlated with the concentrations of limiting nutrients (de Groot et al. 2001, 2003). Larger roots of neighbouring plants correlated with larger Melaleuca shoot biomass gains. Had the neighbouring plants been competing for nutrients, the expected effect on Melaleuca would have been the opposite, that is, a decreased nutrient content. This result strengthens the evidence for enhanced nutrient uptake, made possible by the combination of efficient nutrient-mining as well as nutrient-scavenging neighbours in nutrient-poor soils. In accordance, fertilization removed the positive effect of neighbours, as soil nutrient levels were no longer limiting.

Ectomycorrhizal fungi and cluster–root interactions

Mycorrhizal colonization levels and the type of mycorrhiza formed on Melaleuca were influenced by the neighbouring plants. The presence of the obligate AMF host V. nitens favoured the dominance of AMF on the focal plant Melaleuca. Similarly, in the presence of EMF host Eucalyptus, Melaleuca formed more ectomycorrhizas. These results show that neighbouring plants also promote greater colonization levels, likely via mycorrhizal networks (Simard et al. 2012), and influence the type of mycorrhiza that predominates in plant communities as reported in other ecosystems (van der Heijden & Horton 2009). We note that AMF may have suppressive effects on EMF formation as suggested in recent studies with Australian plants (Janos et al. 2013).

Arbuscular mycorrhizal fungi are well known for enhancing P uptake in plants, up to 90% in some cases (van der Heijden, Bardgett & van Straalen 2008). This is one mechanism by which AMF can enhance plant productivity, especially when P is limiting such as in nutrient-poor soils of south-western Australia. However, our results point to enhanced plant productivity mediated by EMF in the presence of a P-mining cluster-rooted plant, most likely because the soils are more severely P impoverished than the nutrient-poor soils where AMF tend to have a positive effect (Lambers & Teste 2013; Lambers et al. In press).

Ectomycorrhizal fungi have traditionally been viewed as key symbionts in maintaining plant productivity in boreal and temperate forests (Smith & Read 2008). These ecosystems are usually poor in N where EMF can scavenge vast amounts of organic compounds in soil using extracellular enzymes (Read & Perez-Moreno 2003). Our study points to the underestimated importance of EMF in P acquisition, as suggested before by others (Cairney 2011). The importance of EMF in uptake of P is noticeable when P is limiting plant growth (Plassard & Dell 2010). We propose that EMF scavenges released P in the vicinity of cluster roots where large amounts of P can be mobilized (Lambers et al. 2012). Thus, our findings provide support for the expectations that possible facilitative root interactions prevail in nutrient-poor soils (Hauggaard-Nielsen & Jensen 2005). However, confirmation through rigorous experiments using tracers and in vivo functional visualization techniques is warranted (Courty et al. 2010).

Root intermingling and competition

Root competition has traditionally been thought to commonly occur when plants grow together in close proximity in soils with nutrient-rich patches (de Kroon, Mommer & Nishiwaki 2003). In nutrient-poor soils, root competition should also be common; however, here a plant's ability to intermingle with roots of neighbouring plants can have net positive effects on growth (Wilson 1989; Lambers & Teste 2013). The greater growth of Melaleuca growing beside Eucalyptus and Banksia coincided with the greatest amount of root intermingling compared only with one of the other plant assemblies only under nutrient-poor soils. We suggest that, in nutrient-poor soils, hotspots of nutrient mobilization and mineralization are found around certain neighbouring plant roots and thus promote root proliferation analogous to nutrient patches. In fertilized soils, root intermingling declined and showed no pattern with neighbour identity. With fertilization, plant diversity and coexistence decreased in plant mixtures where only root competition occurred (Rajaniemi, Allison & Goldberg 2003).

Root competition is predicted to be critical to plant productivity in mixtures especially when light is abundant but nutrients are limiting (Wilson 1988). Positive plant–plant interactions can occur in crop plant assemblies, where one plant species takes up more nutrients than the other two neighbouring plants (Wilson 1989). In this study, light and water were not limiting; thus, it appeared advantageous to explore larger volumes of soil laterally despite the presence of neighbouring plants' root systems. Net positive effects of this nature have been reported in dry relatively nutrient-poor forests where greater access to mycorrhizal networks and roots promoted survival of forest seedlings (Teste et al. 2009).

The observation that the greatest stimulation of Melaleuca growth was found in the presence of mesh barriers between neighbouring plants suggests a positive effect of spatial separation of roots. However, Melaleuca shoot growth was greater, rather than smaller when larger amounts of neighbouring plant roots intermingled (Fig. 7), indicating that sharing rhizospheres led to a positive net benefit. Root proliferation in plant mixtures should follow an evolutionarily stable strategy, where the overall fitness and productivity of the plant is maximized. This strategy entails the proliferation of roots in unoccupied soil first, then intermingling with interspecific neighbours, before exploring soil already occupied by a plant's own roots (Gersani et al. 2001). Root proliferation is typically affected by local nutrient enrichment (Fransen et al. 1999), and our results suggest local enrichment is likely in interspecific rhizosphere soil in nutrient-poor soils. Thus, our study points to simultaneous proliferation of roots in unoccupied soil and in soil occupied by different species.

Concluding remarks

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Concluding remarks
  8. Acknowledgements
  9. References
  10. Supporting Information

A novel microcosm pot experiment that allowed four plant species possessing different nutrient-acquiring strategies to grow with root interactions was conducted. We provide firm evidence for enhanced nutrient uptake in the plant assembly that had an EMF host and a cluster–root forming plant. Promoted growth matched increased acquisition of macronutrients P and Ca, and of micronutrients Fe, Mn and B. The data suggest a synergistic effect, encompassing the effective EMF hyphal scavenging of limiting nutrients mobilized by organic anions of the cluster roots. Mycorrhizal networks appeared important for greater colonization and the dominance of the type of mycorrhiza forming on the focal plant Melaleuca. While in our experiment, each of the different nutrient-acquisition strategies was represented by a single species, we minimized potential idiosyncratic results by choosing plant species that are widespread, abundant, representative, woody and do not differ in other key confounding traits. The facilitative and competitive mechanisms that enable coexistence go far beyond the traditional view that plants interact mainly through resource depletion (Schenk 2006). Further research on root interactions amongst plants with different nutrient-acquiring strategies, in the context of nutrient-poor soils, will provide valuable insights into how root competition or facilitative root interactions can shape plant communities, promote diversification and coevolution of root symbionts regulating these interactions. As such it enhances our understanding of the ecophysiological functioning of biodiversity hotspots in nutrient-impoverished landscapes.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Concluding remarks
  8. Acknowledgements
  9. References
  10. Supporting Information

We thank Raymond Scott at the UWA Combined Workshop for help in designing and building the microcosm pots. Dr John Koch at the Alcoa Huntly mine with Sonja Jakob and Michael Blair at the UWA Shenton Park Field Station facilitated the collection of the soil. We are grateful to Osmarina Alves Marinho, Ana Luíza Muler and Gregory Cawthray for help during the laboratory work. Funding was provided by the Australian Research Council with a Discovery Project to HL, EJV and KWD.

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  4. Materials and methods
  5. Results
  6. Discussion
  7. Concluding remarks
  8. Acknowledgements
  9. References
  10. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Concluding remarks
  8. Acknowledgements
  9. References
  10. Supporting Information
FilenameFormatSizeDescription
fec12270-sup-0001-LaySummary.pdfPDF document209KLaySummary
fec12270-sup-0002-TableS1-S2-FigS1-S4.docxWord document709K

Fig. S1. Height increment of Melaleuca preissiana (Mp) as the focal plant in a microcosm with combinations of Em: Eucalyptus marginata, Vn: Verticordia nitens and Bm: Banksia menziesii.

Fig. S2. Shoot calcium (Ca), iron (Fe) and boron (B) content of Melaleuca preissiana as the focal plant in a microcosm with combinations of Em: Eucalyptus marginata, Vn: Verticordia nitens and Bm: Banksia menziesii.

Fig. S3. Regression analyses of the focal plant (Melaleuca preissiana) net shoot growth with the final root biomass of neighbouring plants.

Fig. S4. Photographs of Mp: Melaleuca preissiana, Em: Eucalyptus marginata, Vn: Verticordia nitens and Bm: Banksia menziesii roots highlighting the different morphologies of the different nutrient-acquisition strategies present.

Table S1. Analysis of variance (ANOVA) table for the net shoot biomass gain of Melaleuca preissiana as a focal plant in fertilized pots.

Table S2. Analysis of variance (ANOVA) table for the net root biomass gain of Melaleuca preissiana as a focal plant in fertilized pots.

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