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Nutritional ecology of arbuscular mycorrhizal fungi
fungal ecology 3 (2010) 267–273
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/funeco
Mini-review
Nutritional ecology of arbuscular mycorrhizal fungi A. HODGE*, T. HELGASON, A.H. FITTER Department of Biology, Area 14, PO Box 373, University of York, York YO10 5YW, UK
article info
abstract
Article history:
Despite their large role in ecosystems and plant nutrition, our knowledge of the nutritional
Received 7 August 2009
ecology of the fungi involved in the arbuscular mycorrhizal symbiosis, the Glomeromycota,
Revision received 1 February 2010
is poor. We briefly describe the mechanisms that underlie the fluxes of the three major
Accepted 3 February 2010
elements (C, N and P) and outline a model for the interchange of these between the
Available online 30 March 2010
partners. This model is consistent with data from physiological, ecological and taxonomic
Corresponding editor: John Cairney
studies and allows a new and necessary focus on the nutritional requirements of the fungus itself, separately from its role in the symbiosis. There is an urgent need for new
Keywords:
studies to identify the sources of nutrients such as N and P that AM fungi (AMF) use for
Carbon
their own growth and to elucidate the mechanisms that control the transfer of these to the
Evolution
plant in relation to fungal demand.
Glomeromycota
ª 2010 Elsevier Ltd and The British Mycological Society. All rights reserved.
Mycelium Mycorrhiza Nitrogen Nutrition Phosphorus
Introduction Traditionally the arbuscular mycorrhizal (AM) symbiosis is viewed as a classic mutualism, an interaction in which both partners benefit. The fungi appear to acquire their entire carbon supply from the plant, and although colonisation of roots by AM fungi (AMF) can confer a wide range of benefits to the plant (Newsham et al. 1995), the most widely cited benefit is that of enhanced phosphorus (P) acquisition. The fungal hyphae can explore a large volume of soil and acquire P beyond the phosphate depletion zone that rapidly builds up around the root surface at a much smaller carbon cost than is possible by root growth (Harley 1989): this economy probably underlies the evolution of the symbiosis. The fossil record of the AM fungal
phylum Glomeromycota goes back to the Devonian as a symbiosis (Remy et al. 1994) and to the Ordovician as spores (Redecker et al. 2000); they thus have a contemporaneous origin with the land flora. The first land plants had rhizomes and rhizoids, but no root systems. Acquisition of poorly mobile phosphate ions was therefore a major problem and fossil evidence reveals that these early plants had fungal structures strikingly similar to modern AM structures of the ‘Arum-type’ in their rhizomes; it is not a big leap to the assumption that they performed the same function then as now, and were responsible for plant uptake of P (Helgason & Fitter 2009). That enhanced plant P nutrition is still a major outcome of the AM symbiosis demonstrates that while root systems have become larger and more complex, P acquisition is still a major challenge for most plants.
* Corresponding author. Tel.: þ44 1904 328562. E-mail address: [email protected] (A. Hodge). 1754-5048/$ – see front matter ª 2010 Elsevier Ltd and The British Mycological Society. All rights reserved. doi:10.1016/j.funeco.2010.02.002
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The morphology of AMF colonisation can vary giving rise to the so-called ‘Arum-’ or ‘Paris-type’ mycorrhizas. In the Arum type inter-cellular AMF hyphae spread within the root cortex. Short side branches form which penetrate the root cortical cell walls and branch extensively to give the characteristic ‘arbuscule’ structure. In the Paris type there is little inter-cellular growth but, instead, extensive intracellular coiled hyphae which spread directly from cell to cell and from which arbuscules may develop. Much less is known about the Paris type than the Arum type and it is the latter that forms in the roots of most crop plant species (Smith & Read 2008). Around two-thirds of all plants form the AM symbiosis but, for a group with such ecological importance, our knowledge of the biology of the Glomeromycota is comparatively poor. Recent studies show that there is considerable genetic and phenotypic variation among AM fungal isolates (Koch et al. 2004; Croll et al. 2008), and although sexual stages have never been observed, genetic evidence suggests that recombination may occur (Gandolfi et al. 2003; Croll & Sanders 2009). These exciting new studies suggest that a greater understanding of AM fungal population structure, differentiation, dispersal and persistence is not far away; much nevertheless remains to be done. Critically for the purpose of this review we do not know why they are obligate symbionts and cannot be grown in the absence of live plant tissue, nor the most basic details of their physiology and especially what controls the fluxes of nutrients between plant and fungus in the symbiosis. This review examines the current knowledge of the nutritional ecology of AM fungi.
Nutrient fluxes in the AM symbiosis Carbon The major fluxes in the AM symbiosis appear to be of C from plant to fungus and of P, and possibly N, from fungus to plant. Reverse C movement – from fungus to plant – appears only to occur in special cases where the plant has an unusually restricted C supply, most notably in achlorophyllous plants (Bidartondo et al. 2002). In virtually all other cases, apparent plant-to-plant movement of C is best explained as the AM fungus moving C from the intra-radical mycelium in one root system to the same mycelium within another root; the carbon almost always remains in the roots and is retained in the intra-radical fungal structures (Robinson & Fitter 1999; Voets et al. 2008). The mechanisms of these fluxes are not yet well understood. Even the location of the carbon flux is obscure, with the best evidence – from activity of ATPases – suggesting that it occurs in the Arum type at the inter-cellular hyphae (Gianinazzi-Pearson et al. 1991). A model for transport of C from intra- to extra-radical hyphae has been proposed (Bago et al. 2003) and a hexose transporter (GpMST1) has been identified in the fungus Geosiphon pyriforme, a nonmycorrhizal member of the Glomeromycota (Schu¨bler et al. 2006). The identity of the C transporters in mycorrhizal taxa will soon become apparent as genome information is published (Martin et al. 2008).
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Phosphate The phosphate flux is better characterised. Phosphate is taken up by high-affinity phosphate transporters in the extra-radical mycelium (Harrison & van Buuren 1995). Phosphate is probably transported within the fungus as polyphosphate (polyP), and once in the intra-radical hyphae the long chains are hydrolysed, facilitating transfer to the host plant (Harrison 1999; Bago et al. 2002; Ohtomo & Saito 2005). Fungus-to-plant transfer appears to occur principally at the arbuscule interface, although expression of P transporters around Paris type hyphal coils has also been demonstrated (Karandashov et al. 2004). Plant ATPase activity is strongly expressed at the periarbuscular membrane (Smith et al. 2009) and phosphate accumulation as polyP strongly correlated with AM colonisation (Ohtomo & Saito 2005). Most importantly, a subfamily (subfamily 1 under the family Pht1) of plant phosphate transporters is now known that is expressed only in colonised plants; the first of these was in Solanum tuberosum (StPT4; Rausch et al. 2001), and they have subsequently been identified in several other taxa (Javot et al. 2007). Acquisition of P via the symbiotic pathway downregulates direct P uptake by the plant (Smith et al. 2004, 2009).
Nitrogen In contrast to P, fewer studies have considered a role for AMF in N acquisition, because the greater mobility of ammonium and especially nitrate ions in soil, compared to phosphate, led to the assumption that little benefit was likely to plants from enhanced N uptake. AMF can certainly transport N to roots: AM extra-radical mycelium (ERM) exposed to 15N-labelled NO 3 or NHþ 4 became highly labelled and this N was subsequently translocated to the roots (Govindarajulu et al. 2005), confirming earlier work (Tobar et al. 1994; Johansen et al. 1996; Ma¨der et al. 2000). N is translocated in the hyphae as arginine but probably broken down to urea and ultimately transferred to the plant as NHþ 4 with the resulting C skeletons from arginine breakdown being re-incorporated into the fungal C pools (Bago et al. 2001; Govindarajulu et al. 2005). A plant ammonium transporter (AMT) has recently been identified in Lotus japonicus which is mycorrhiza-specific and preferentially expressed in arbusculated cells (Guether et al. 2009a, b), and up-regulation of an ammonium transporter in Medicago truncatula has also been found (Gomez et al. 2009). Moreover, Leigh et al. (2009) demonstrated that a fifth of plant N could be derived from AM fungal transfer when only the fungus had access to a compartment containing an organic N source. However, the role played by AM fungi in N acquisition from organic N sources, the dominant form of N in most soils, remains controversial (but see Whiteside et al. 2009), especially when both roots and AM hyphae have access to the same N source (Hodge et al. 2000a; Hodge 2003a).
Plant–fungus reciprocity: who drives whom? These fluxes underlie the operation of the symbiosis, but we do not know how the exchange is managed. Is there some reciprocity between the C supplied by the plant and the P (or N)
Nutritional ecology of AM fungi
supplied by the fungus? If so, there will have been powerful selection on the operation of this mechanism, with potentially conflicting pressures on the two partners. There are apparently 1 000 times more species of plant involved in the AM symbiosis than of fungi (the ratio of described species is w2 105:2 102). Even if the number of fungal species is a serious underestimate, most fungi must be able to colonise many plant species, a conclusion supported by the lack of specificity of all fungi known in culture. Nevertheless there is great variation in the effectiveness of particular pairings of plant and fungal taxa (van der Heijden et al. 1998; Pringle & Bever 2002; Klironomos 2003; Helgason et al. 2007), suggesting that some fungi are better partners than others. If that is the case, plants will have been under powerful selection to discriminate among fungal partners on the basis of symbiotic effectiveness. One possible mechanism for that would be a set of molecular signals specific to each fungus. The establishment of the symbiosis involves just such a recognition process (Akiyama & Hayashi 2006), but it appears to be a general one, that has been exploited by symbioses that evolved subsequently (root nodules with Rhizobium and the parasitic plant Striga). However, a recognition-based system depends on honest signals and is very vulnerable to cheats – taxa that copy the signals but offer no benefits to the partner. The remarkable durability of the symbiosis – over 400 MYr – suggests that it is resistant to invasion by such cheats, and an alternative mechanism for the regulation of these nutrient fluxes provides a more nearly cheat-proof model. This model relies on the fundamental idea that the plant will transfer C to the fungus only in direct response to the transfer of N or P. Achieving that does not require a direct linkage of the exchange mechanism, but relies on known physiology and the spatial pattern of fungal colonisation and root behaviour. Briefly, the main elements of the model (Fitter 2006; Helgason & Fitter 2009) are: 1. AM fungi colonise roots in discrete patches. If the fungus is successfully to acquire C from the root, it must generate the C flux at that scale. 2. Roots can detect heterogeneity of nutrient supply at a fine scale and respond by local proliferation (Drew 1975). 3. When an AM fungus transfers P (or possibly N) across the arbuscule membranes, there will be a local increase in phosphate (or ammonium) concentration in the root. The plant will not be able to distinguish that from the increase that results from enhanced epidermal uptake and will respond by differential transport of hexoses to the site of increased uptake. 4. Some of those hexoses will leak into the apoplast and be acquired by the inter-cellular hyphae. Smith et al. (2009) have suggested the Fitter (2006) model may be an over-simplification because it does not allow for ‘cheating’ in individual fungal–plant interactions, which is held to exist because of growth depressions of the host observed in some AM interactions (Johnson et al. 1997; Klironomos 2003). However, AMF are multi-functional and the benefit to the host may not always be nutritional, expressed in simple culture conditions or obvious (see Newsham et al. 1995). A strict demonstration of cheating is
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extremely hard to achieve, and the mere demonstration of a growth depression is insufficient, because of potential unmeasured responses that would be beneficial in realistic environments. It is notable that AMF may transfer P to the host in the absence of any growth response (Smith et al. 2009); the same argument would describe this as the plant cheating the fungus. The manner in which the majority of mycorrhizal research is conducted (i.e. a single fungus and a single plant with the plant generally trying to establish itself plus the fungal mycelium de novo at the same time) is artificial. Under natural conditions, the seedling would plug into an established AM common mycelial network (CMN): the carbon burden would therefore initially be less. Smith et al. (2009) have also pointed out that cortical colonisation is not essential for C transfer. In Paris type colonisation there is no inter-cellular phase, suggesting that transfer must be intracellular; similarly, colonisation of the rmc mutant of tomato occurs only in the epidermis and hypodermis but can still result in the fungus producing extraradical mycelium and even spores (Manjarrez et al. 2008). It is clear therefore, that C transfer cannot occur exclusively at the cortical inter-cellular interface but instead must occur at numerous sites. In practice, the precise location of C transfer is not a key feature of the model: what is required is that P (or N) release in the arbuscule stimulates a carbon flux to the colonised areas of the root, a phenomenon that has been experimentally demonstrated (Javot et al. 2007).
Nutrient acquisition by AM fungi Perhaps because of their key role in plant P acquisition, AM fungi have largely been viewed as extensions of the plant root system. That view ignores the nutritional needs of the fungus and, importantly, that those nutritional needs may be in conflict with the plant’s when resource availability is low. Unlike other mycorrhizal associations, where at least some of the fungi involved can be grown in pure culture and we can measure the capability of the fungus acting alone to decompose organic materials and take up the products of decomposition (Hodge et al. 1995; Read & Perez-Moreno 2003), it is currently impossible to culture AM fungi in the absence of a host plant. Despite pleas for a more ‘mycocentric’ approach (Fitter et al. 2000; Alberton et al. 2005; Southworth et al. 2005; Alberton & Kuyper 2009), this lacuna has fuelled a view that what is good for the plant must surely benefit the fungus and that the fungus operates in such a way as to benefit its host at all times. In common with plant roots (Hodge 2009), AM fungi proliferate hyphae in nutrient-rich patches of organic matter under both controlled and field conditions (Mosse 1959; Nicolson 1959; St John et al. 1983; Joner & Jakobsen 1995; Hodge et al. 2001; Cavagnaro et al. 2005), a behaviour generally viewed as a foraging response in a heterogeneous environment. AMF, however, are not saprotrophic (Smith & Read 2008) and therefore are reliant on saprotrophic microorganisms to decompose organic matter and release inorganic ions for capture by AM hyphae (but see Whiteside et al. 2009; Hawkins et al. 2000). Over short time scales, plant roots compete weakly with microbes for the released resources
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(Kaye & Hart 1997; Hodge et al. 2000b) and root proliferation is more likely to affect inter-plant competition (Hodge 2009). AMF, however, must also compete with other microbes and their lack of saprotrophic capability should set them at a similar disadvantage. However, the presence of AMF in an organic patch can enhance its decomposition (Hodge et al. 2001; Atul-Nayyar et al. 2009). The mechanism for this enhancement is unknown, but AMF hyphae may have a direct influence on other microorganisms in the ‘hyphosphere’ (Toljander et al. 2007). Some of the carbon in the AMF hyphae may be exuded or secreted, which would encourage bacterial growth in a similar way to the well studied ‘rhizosphere’ effect (Ravnskov et al. 1999). The external AM mycelium phase is the fungal phase which is in contact with the soil and thus responsible for nutrient acquisition and transport to the internal mycelium inside the root before any transfer to the plant occurs. In return, triacylglycerides can be transported from the internal to the external mycelium phase to support the glyoxyl cycle for metabolic activity (Pfeffer et al. 1999; Lammers et al. 2001). However, despite the obvious importance of the external mycelium in nutrient acquisition, few fungal transporters have been characterised. These include a phosphate (Harrison & van Buuren 1995), an ammonium (Lo´pez-Pedrosa et al. 2006) and a putative zinc transporter (Gonza´lez-Guerrero et al. 2005). In addition, an aquaporin (water channel proteins) gene, GintAQP1, has been discovered in the external mycelium of Glomus intraradices which showed increased expression in parts of the AMF mycelium not experiencing osmotic stress compared to parts that were (Aroca et al. 2009). This suggests communication between the different parts of the mycelium subject to the differing external conditions, a behaviour that is well established in roots (Hodge 2009). In the case of ammonium a NHþ 4 transporter gene (GintAMT1), with high sequence similarity to NHþ 4 transporters characterised in other fungi, has been identified in the extraradical mycelium of G. intraradices (Lo´pez-Pedrosa et al. 2006). GintAMT1 was up-regulated after the addition of low NHþ 4 concentrations to the media but down-regulated when higher NHþ 4 concentrations were added, suggesting that it is a high´ pez-Pedrosa et al. 2006) and that affinity NHþ 4 transporter (Lo lower affinity NHþ 4 transporters have yet to be identified. A high-affinity phosphate transporter (GvPT ) has also been cloned from Glomus versiforme (Harrison & van Buuren 1995). The expression and regulation of GiPT, a homolog of GvPT, from G. intraradices was regulated in the external mycelium in response to phosphate concentration of the external environment. Further, the phosphate status of the mycorrhizal root influenced both phosphate uptake and GiPT expression in the ERM (Maldonado-Mendoza et al. 2001). This suggests the ERM of AMF can detect and show physiological plasticity in response to the nutrient status of their environment and host. Morphological responses of the external mycelium of AMF in response to nutrient status of their environment have also been demonstrated (Bago et al. 2004; Leigh et al. 2009) as have differing substrate colonisation strategies among AMF genera (Cano & Bago 2005) albeit under rather artificial conditions. Bago et al. (2004) reported that under low nutrient conditions the ERM developmental pattern was one that allowed both exploration and exploitation of the growth medium: runner
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hyphae radially extended the fungal colony from which branched absorbing structures developed at regular intervals or from which spores developed in older parts. The life-spans of these hyphal structures also appear to differ (Staddon et al. 2003); thicker hyphae probably live longer and determine the development of the hyphal network, analogous to plant root system development. How the external hyphae sense their environment in order to display the substantial hyphal proliferation in organic rich patches is unknown, but may involve the nutrient ions acting as a direct signal as has been demonstrated for nitrate and root proliferation (Zhang & Forde 1998). Application of proteomic and candidate target gene approaches specifically applied to the external mycelium offer a way forward to further determine the functioning of this key AM fungal phase in the environment (see Recorbet et al. 2009; Gamper et al. 2010) but must be carried out under ecologically meaningful conditions if the link with function is to be established. N capture by AM fungi was previously believed to have little ecological relevance. An important and influential article by Read (1991) argued that AM associations tend to dominate in systems where nitrification is favoured and the main form of inorganic N will consequently be nitrate. As NO 3 , unlike phosphate, is highly mobile in soils and depletion zones around roots can be measured in centimetres rather then millimetres, plants should not require AM fungi in order to enhance capture of NO 3 . However, it is now clear that the external hyphae of AMF take up inorganic N as both NO 3 and NHþ 4 (Bago et al. 1996; Govindarajulu et al. 2005; Jin et al. 2005) and organic N as amino acids (Hawkins et al. 2000) and transfer some – sometimes a large fraction – to the plant. The ecological significance of this transfer is still uncertain. NHþ 4 may be the preferred fungal N source under most circumstances (Hawkins et al. 2000; Read & Perez-Moreno 2003; but see Azco´n et al. 1996). N transfer to the plant may also be higher when NHþ 4 , rather than NO3 , is supplied to the AMF hyphae (Tanaka & Yano 2005) even though N is likely transferred from the external to the internal AM hyphae as arginine (Govindarajulu et al. 2005; Cruz et al. 2007) before transfer to the plant as NHþ 4 (Gomez et al. 2009; Guether et al. uptake may be less energetically expensive for 2009a). NHþ 4 þ the fungus as NO 3 first has to be reduced to NH4 prior to incorporation into amino acids. While these N sources must be important for fungal nutrition, the contribution to plant nutrition is controversial (see Read & Perez-Moreno 2003; Leigh et al. 2009) and may depend on the relative N requirements of plant and fungus. In some systems, such as the acidic organic soils frequent in many tropical areas (Moyersoen et al. 2001), AMF NHþ 4 capture and subsequent transfer to the host plant may be of considerable ecological significance. When mycorrhizal roots have access to patches of organic material in soil the AMF appear not to respond (Hodge 2001, 2003b); hyphal proliferation occurs only when the AMF alone have access to the patch (Joner & Jakobsen 1995; Hodge et al. 2001). One remarkable result in Hodge et al. (2001) was that the fungus grew preferentially into a patch of organic matter rather than towards a new, uncolonised host plant. If the fungus gains all its carbon from the host, this implies that the fungus gets an alternative benefit from the patch greater than
Nutritional ecology of AM fungi
that it can gain from a new carbon source. In fact there is good evidence that AMF obtain a growth benefit from organic matter in soil. Leigh et al. (2009) found increased hyphal growth in the ‘plant’ compartment (i.e. that in which both plant and fungus grew) when the fungus was allowed to explore a second, ‘hyphal’ compartment that contained an organic matter patch. Thus, what seems to have been overlooked in the debate over AM fungal responses to organic matter is the possibility that it represents a major N source for the fungus itself. Indeed, there are no studies that directly address the question of the sources of fungal N under realistic conditions. Those that show transfer of N by the fungus to the plant may be revealing a consequence of over-supply of N to the fungus. The demonstration that the supply of organic N compounds can elicit a transcriptional response in G. intraradices (Cappellazzo et al. 2007) adds weight to this suggestion.
Conclusions An increased acceptance of the independent but interacting roles of plant and fungus in nutrient transfers allows a new and necessary focus on the nutritional needs of the fungus itself. All fungi have a high nitrogen content and hence potentially a high N demand. Indeed because AMF are among the most abundant fungi on earth, their role in global N and P cycles would repay close attention. There is abundant evidence that AMF can acquire N (and presumably also P) from decomposing organic material and transfer it to the plant. The N and P transfer to the plant may, if the model proposed above is correct, be a consequence of the fungal demand for nutrients, with both host plant and fungus evolving transporters to take advantage of localised increases in nutrients. However, the mechanisms by which these fungi actively forage in soil for both N and P remain unclear. What is certain is that we need to pay much more attention to the biology and ecology of the extra-radical phase of these fungi if we are to understand how they operate in soil and the roles that they play in ecosystems.
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Mini-review
Nutritional ecology of arbuscular mycorrhizal fungi A. HODGE*, T. HELGASON, A.H. FITTER Department of Biology, Area 14, PO Box 373, University of York, York YO10 5YW, UK
article info
abstract
Article history:
Despite their large role in ecosystems and plant nutrition, our knowledge of the nutritional
Received 7 August 2009
ecology of the fungi involved in the arbuscular mycorrhizal symbiosis, the Glomeromycota,
Revision received 1 February 2010
is poor. We briefly describe the mechanisms that underlie the fluxes of the three major
Accepted 3 February 2010
elements (C, N and P) and outline a model for the interchange of these between the
Available online 30 March 2010
partners. This model is consistent with data from physiological, ecological and taxonomic
Corresponding editor: John Cairney
studies and allows a new and necessary focus on the nutritional requirements of the fungus itself, separately from its role in the symbiosis. There is an urgent need for new
Keywords:
studies to identify the sources of nutrients such as N and P that AM fungi (AMF) use for
Carbon
their own growth and to elucidate the mechanisms that control the transfer of these to the
Evolution
plant in relation to fungal demand.
Glomeromycota
ª 2010 Elsevier Ltd and The British Mycological Society. All rights reserved.
Mycelium Mycorrhiza Nitrogen Nutrition Phosphorus
Introduction Traditionally the arbuscular mycorrhizal (AM) symbiosis is viewed as a classic mutualism, an interaction in which both partners benefit. The fungi appear to acquire their entire carbon supply from the plant, and although colonisation of roots by AM fungi (AMF) can confer a wide range of benefits to the plant (Newsham et al. 1995), the most widely cited benefit is that of enhanced phosphorus (P) acquisition. The fungal hyphae can explore a large volume of soil and acquire P beyond the phosphate depletion zone that rapidly builds up around the root surface at a much smaller carbon cost than is possible by root growth (Harley 1989): this economy probably underlies the evolution of the symbiosis. The fossil record of the AM fungal
phylum Glomeromycota goes back to the Devonian as a symbiosis (Remy et al. 1994) and to the Ordovician as spores (Redecker et al. 2000); they thus have a contemporaneous origin with the land flora. The first land plants had rhizomes and rhizoids, but no root systems. Acquisition of poorly mobile phosphate ions was therefore a major problem and fossil evidence reveals that these early plants had fungal structures strikingly similar to modern AM structures of the ‘Arum-type’ in their rhizomes; it is not a big leap to the assumption that they performed the same function then as now, and were responsible for plant uptake of P (Helgason & Fitter 2009). That enhanced plant P nutrition is still a major outcome of the AM symbiosis demonstrates that while root systems have become larger and more complex, P acquisition is still a major challenge for most plants.
* Corresponding author. Tel.: þ44 1904 328562. E-mail address: [email protected] (A. Hodge). 1754-5048/$ – see front matter ª 2010 Elsevier Ltd and The British Mycological Society. All rights reserved. doi:10.1016/j.funeco.2010.02.002
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The morphology of AMF colonisation can vary giving rise to the so-called ‘Arum-’ or ‘Paris-type’ mycorrhizas. In the Arum type inter-cellular AMF hyphae spread within the root cortex. Short side branches form which penetrate the root cortical cell walls and branch extensively to give the characteristic ‘arbuscule’ structure. In the Paris type there is little inter-cellular growth but, instead, extensive intracellular coiled hyphae which spread directly from cell to cell and from which arbuscules may develop. Much less is known about the Paris type than the Arum type and it is the latter that forms in the roots of most crop plant species (Smith & Read 2008). Around two-thirds of all plants form the AM symbiosis but, for a group with such ecological importance, our knowledge of the biology of the Glomeromycota is comparatively poor. Recent studies show that there is considerable genetic and phenotypic variation among AM fungal isolates (Koch et al. 2004; Croll et al. 2008), and although sexual stages have never been observed, genetic evidence suggests that recombination may occur (Gandolfi et al. 2003; Croll & Sanders 2009). These exciting new studies suggest that a greater understanding of AM fungal population structure, differentiation, dispersal and persistence is not far away; much nevertheless remains to be done. Critically for the purpose of this review we do not know why they are obligate symbionts and cannot be grown in the absence of live plant tissue, nor the most basic details of their physiology and especially what controls the fluxes of nutrients between plant and fungus in the symbiosis. This review examines the current knowledge of the nutritional ecology of AM fungi.
Nutrient fluxes in the AM symbiosis Carbon The major fluxes in the AM symbiosis appear to be of C from plant to fungus and of P, and possibly N, from fungus to plant. Reverse C movement – from fungus to plant – appears only to occur in special cases where the plant has an unusually restricted C supply, most notably in achlorophyllous plants (Bidartondo et al. 2002). In virtually all other cases, apparent plant-to-plant movement of C is best explained as the AM fungus moving C from the intra-radical mycelium in one root system to the same mycelium within another root; the carbon almost always remains in the roots and is retained in the intra-radical fungal structures (Robinson & Fitter 1999; Voets et al. 2008). The mechanisms of these fluxes are not yet well understood. Even the location of the carbon flux is obscure, with the best evidence – from activity of ATPases – suggesting that it occurs in the Arum type at the inter-cellular hyphae (Gianinazzi-Pearson et al. 1991). A model for transport of C from intra- to extra-radical hyphae has been proposed (Bago et al. 2003) and a hexose transporter (GpMST1) has been identified in the fungus Geosiphon pyriforme, a nonmycorrhizal member of the Glomeromycota (Schu¨bler et al. 2006). The identity of the C transporters in mycorrhizal taxa will soon become apparent as genome information is published (Martin et al. 2008).
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Phosphate The phosphate flux is better characterised. Phosphate is taken up by high-affinity phosphate transporters in the extra-radical mycelium (Harrison & van Buuren 1995). Phosphate is probably transported within the fungus as polyphosphate (polyP), and once in the intra-radical hyphae the long chains are hydrolysed, facilitating transfer to the host plant (Harrison 1999; Bago et al. 2002; Ohtomo & Saito 2005). Fungus-to-plant transfer appears to occur principally at the arbuscule interface, although expression of P transporters around Paris type hyphal coils has also been demonstrated (Karandashov et al. 2004). Plant ATPase activity is strongly expressed at the periarbuscular membrane (Smith et al. 2009) and phosphate accumulation as polyP strongly correlated with AM colonisation (Ohtomo & Saito 2005). Most importantly, a subfamily (subfamily 1 under the family Pht1) of plant phosphate transporters is now known that is expressed only in colonised plants; the first of these was in Solanum tuberosum (StPT4; Rausch et al. 2001), and they have subsequently been identified in several other taxa (Javot et al. 2007). Acquisition of P via the symbiotic pathway downregulates direct P uptake by the plant (Smith et al. 2004, 2009).
Nitrogen In contrast to P, fewer studies have considered a role for AMF in N acquisition, because the greater mobility of ammonium and especially nitrate ions in soil, compared to phosphate, led to the assumption that little benefit was likely to plants from enhanced N uptake. AMF can certainly transport N to roots: AM extra-radical mycelium (ERM) exposed to 15N-labelled NO 3 or NHþ 4 became highly labelled and this N was subsequently translocated to the roots (Govindarajulu et al. 2005), confirming earlier work (Tobar et al. 1994; Johansen et al. 1996; Ma¨der et al. 2000). N is translocated in the hyphae as arginine but probably broken down to urea and ultimately transferred to the plant as NHþ 4 with the resulting C skeletons from arginine breakdown being re-incorporated into the fungal C pools (Bago et al. 2001; Govindarajulu et al. 2005). A plant ammonium transporter (AMT) has recently been identified in Lotus japonicus which is mycorrhiza-specific and preferentially expressed in arbusculated cells (Guether et al. 2009a, b), and up-regulation of an ammonium transporter in Medicago truncatula has also been found (Gomez et al. 2009). Moreover, Leigh et al. (2009) demonstrated that a fifth of plant N could be derived from AM fungal transfer when only the fungus had access to a compartment containing an organic N source. However, the role played by AM fungi in N acquisition from organic N sources, the dominant form of N in most soils, remains controversial (but see Whiteside et al. 2009), especially when both roots and AM hyphae have access to the same N source (Hodge et al. 2000a; Hodge 2003a).
Plant–fungus reciprocity: who drives whom? These fluxes underlie the operation of the symbiosis, but we do not know how the exchange is managed. Is there some reciprocity between the C supplied by the plant and the P (or N)
Nutritional ecology of AM fungi
supplied by the fungus? If so, there will have been powerful selection on the operation of this mechanism, with potentially conflicting pressures on the two partners. There are apparently 1 000 times more species of plant involved in the AM symbiosis than of fungi (the ratio of described species is w2 105:2 102). Even if the number of fungal species is a serious underestimate, most fungi must be able to colonise many plant species, a conclusion supported by the lack of specificity of all fungi known in culture. Nevertheless there is great variation in the effectiveness of particular pairings of plant and fungal taxa (van der Heijden et al. 1998; Pringle & Bever 2002; Klironomos 2003; Helgason et al. 2007), suggesting that some fungi are better partners than others. If that is the case, plants will have been under powerful selection to discriminate among fungal partners on the basis of symbiotic effectiveness. One possible mechanism for that would be a set of molecular signals specific to each fungus. The establishment of the symbiosis involves just such a recognition process (Akiyama & Hayashi 2006), but it appears to be a general one, that has been exploited by symbioses that evolved subsequently (root nodules with Rhizobium and the parasitic plant Striga). However, a recognition-based system depends on honest signals and is very vulnerable to cheats – taxa that copy the signals but offer no benefits to the partner. The remarkable durability of the symbiosis – over 400 MYr – suggests that it is resistant to invasion by such cheats, and an alternative mechanism for the regulation of these nutrient fluxes provides a more nearly cheat-proof model. This model relies on the fundamental idea that the plant will transfer C to the fungus only in direct response to the transfer of N or P. Achieving that does not require a direct linkage of the exchange mechanism, but relies on known physiology and the spatial pattern of fungal colonisation and root behaviour. Briefly, the main elements of the model (Fitter 2006; Helgason & Fitter 2009) are: 1. AM fungi colonise roots in discrete patches. If the fungus is successfully to acquire C from the root, it must generate the C flux at that scale. 2. Roots can detect heterogeneity of nutrient supply at a fine scale and respond by local proliferation (Drew 1975). 3. When an AM fungus transfers P (or possibly N) across the arbuscule membranes, there will be a local increase in phosphate (or ammonium) concentration in the root. The plant will not be able to distinguish that from the increase that results from enhanced epidermal uptake and will respond by differential transport of hexoses to the site of increased uptake. 4. Some of those hexoses will leak into the apoplast and be acquired by the inter-cellular hyphae. Smith et al. (2009) have suggested the Fitter (2006) model may be an over-simplification because it does not allow for ‘cheating’ in individual fungal–plant interactions, which is held to exist because of growth depressions of the host observed in some AM interactions (Johnson et al. 1997; Klironomos 2003). However, AMF are multi-functional and the benefit to the host may not always be nutritional, expressed in simple culture conditions or obvious (see Newsham et al. 1995). A strict demonstration of cheating is
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extremely hard to achieve, and the mere demonstration of a growth depression is insufficient, because of potential unmeasured responses that would be beneficial in realistic environments. It is notable that AMF may transfer P to the host in the absence of any growth response (Smith et al. 2009); the same argument would describe this as the plant cheating the fungus. The manner in which the majority of mycorrhizal research is conducted (i.e. a single fungus and a single plant with the plant generally trying to establish itself plus the fungal mycelium de novo at the same time) is artificial. Under natural conditions, the seedling would plug into an established AM common mycelial network (CMN): the carbon burden would therefore initially be less. Smith et al. (2009) have also pointed out that cortical colonisation is not essential for C transfer. In Paris type colonisation there is no inter-cellular phase, suggesting that transfer must be intracellular; similarly, colonisation of the rmc mutant of tomato occurs only in the epidermis and hypodermis but can still result in the fungus producing extraradical mycelium and even spores (Manjarrez et al. 2008). It is clear therefore, that C transfer cannot occur exclusively at the cortical inter-cellular interface but instead must occur at numerous sites. In practice, the precise location of C transfer is not a key feature of the model: what is required is that P (or N) release in the arbuscule stimulates a carbon flux to the colonised areas of the root, a phenomenon that has been experimentally demonstrated (Javot et al. 2007).
Nutrient acquisition by AM fungi Perhaps because of their key role in plant P acquisition, AM fungi have largely been viewed as extensions of the plant root system. That view ignores the nutritional needs of the fungus and, importantly, that those nutritional needs may be in conflict with the plant’s when resource availability is low. Unlike other mycorrhizal associations, where at least some of the fungi involved can be grown in pure culture and we can measure the capability of the fungus acting alone to decompose organic materials and take up the products of decomposition (Hodge et al. 1995; Read & Perez-Moreno 2003), it is currently impossible to culture AM fungi in the absence of a host plant. Despite pleas for a more ‘mycocentric’ approach (Fitter et al. 2000; Alberton et al. 2005; Southworth et al. 2005; Alberton & Kuyper 2009), this lacuna has fuelled a view that what is good for the plant must surely benefit the fungus and that the fungus operates in such a way as to benefit its host at all times. In common with plant roots (Hodge 2009), AM fungi proliferate hyphae in nutrient-rich patches of organic matter under both controlled and field conditions (Mosse 1959; Nicolson 1959; St John et al. 1983; Joner & Jakobsen 1995; Hodge et al. 2001; Cavagnaro et al. 2005), a behaviour generally viewed as a foraging response in a heterogeneous environment. AMF, however, are not saprotrophic (Smith & Read 2008) and therefore are reliant on saprotrophic microorganisms to decompose organic matter and release inorganic ions for capture by AM hyphae (but see Whiteside et al. 2009; Hawkins et al. 2000). Over short time scales, plant roots compete weakly with microbes for the released resources
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(Kaye & Hart 1997; Hodge et al. 2000b) and root proliferation is more likely to affect inter-plant competition (Hodge 2009). AMF, however, must also compete with other microbes and their lack of saprotrophic capability should set them at a similar disadvantage. However, the presence of AMF in an organic patch can enhance its decomposition (Hodge et al. 2001; Atul-Nayyar et al. 2009). The mechanism for this enhancement is unknown, but AMF hyphae may have a direct influence on other microorganisms in the ‘hyphosphere’ (Toljander et al. 2007). Some of the carbon in the AMF hyphae may be exuded or secreted, which would encourage bacterial growth in a similar way to the well studied ‘rhizosphere’ effect (Ravnskov et al. 1999). The external AM mycelium phase is the fungal phase which is in contact with the soil and thus responsible for nutrient acquisition and transport to the internal mycelium inside the root before any transfer to the plant occurs. In return, triacylglycerides can be transported from the internal to the external mycelium phase to support the glyoxyl cycle for metabolic activity (Pfeffer et al. 1999; Lammers et al. 2001). However, despite the obvious importance of the external mycelium in nutrient acquisition, few fungal transporters have been characterised. These include a phosphate (Harrison & van Buuren 1995), an ammonium (Lo´pez-Pedrosa et al. 2006) and a putative zinc transporter (Gonza´lez-Guerrero et al. 2005). In addition, an aquaporin (water channel proteins) gene, GintAQP1, has been discovered in the external mycelium of Glomus intraradices which showed increased expression in parts of the AMF mycelium not experiencing osmotic stress compared to parts that were (Aroca et al. 2009). This suggests communication between the different parts of the mycelium subject to the differing external conditions, a behaviour that is well established in roots (Hodge 2009). In the case of ammonium a NHþ 4 transporter gene (GintAMT1), with high sequence similarity to NHþ 4 transporters characterised in other fungi, has been identified in the extraradical mycelium of G. intraradices (Lo´pez-Pedrosa et al. 2006). GintAMT1 was up-regulated after the addition of low NHþ 4 concentrations to the media but down-regulated when higher NHþ 4 concentrations were added, suggesting that it is a high´ pez-Pedrosa et al. 2006) and that affinity NHþ 4 transporter (Lo lower affinity NHþ 4 transporters have yet to be identified. A high-affinity phosphate transporter (GvPT ) has also been cloned from Glomus versiforme (Harrison & van Buuren 1995). The expression and regulation of GiPT, a homolog of GvPT, from G. intraradices was regulated in the external mycelium in response to phosphate concentration of the external environment. Further, the phosphate status of the mycorrhizal root influenced both phosphate uptake and GiPT expression in the ERM (Maldonado-Mendoza et al. 2001). This suggests the ERM of AMF can detect and show physiological plasticity in response to the nutrient status of their environment and host. Morphological responses of the external mycelium of AMF in response to nutrient status of their environment have also been demonstrated (Bago et al. 2004; Leigh et al. 2009) as have differing substrate colonisation strategies among AMF genera (Cano & Bago 2005) albeit under rather artificial conditions. Bago et al. (2004) reported that under low nutrient conditions the ERM developmental pattern was one that allowed both exploration and exploitation of the growth medium: runner
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hyphae radially extended the fungal colony from which branched absorbing structures developed at regular intervals or from which spores developed in older parts. The life-spans of these hyphal structures also appear to differ (Staddon et al. 2003); thicker hyphae probably live longer and determine the development of the hyphal network, analogous to plant root system development. How the external hyphae sense their environment in order to display the substantial hyphal proliferation in organic rich patches is unknown, but may involve the nutrient ions acting as a direct signal as has been demonstrated for nitrate and root proliferation (Zhang & Forde 1998). Application of proteomic and candidate target gene approaches specifically applied to the external mycelium offer a way forward to further determine the functioning of this key AM fungal phase in the environment (see Recorbet et al. 2009; Gamper et al. 2010) but must be carried out under ecologically meaningful conditions if the link with function is to be established. N capture by AM fungi was previously believed to have little ecological relevance. An important and influential article by Read (1991) argued that AM associations tend to dominate in systems where nitrification is favoured and the main form of inorganic N will consequently be nitrate. As NO 3 , unlike phosphate, is highly mobile in soils and depletion zones around roots can be measured in centimetres rather then millimetres, plants should not require AM fungi in order to enhance capture of NO 3 . However, it is now clear that the external hyphae of AMF take up inorganic N as both NO 3 and NHþ 4 (Bago et al. 1996; Govindarajulu et al. 2005; Jin et al. 2005) and organic N as amino acids (Hawkins et al. 2000) and transfer some – sometimes a large fraction – to the plant. The ecological significance of this transfer is still uncertain. NHþ 4 may be the preferred fungal N source under most circumstances (Hawkins et al. 2000; Read & Perez-Moreno 2003; but see Azco´n et al. 1996). N transfer to the plant may also be higher when NHþ 4 , rather than NO3 , is supplied to the AMF hyphae (Tanaka & Yano 2005) even though N is likely transferred from the external to the internal AM hyphae as arginine (Govindarajulu et al. 2005; Cruz et al. 2007) before transfer to the plant as NHþ 4 (Gomez et al. 2009; Guether et al. uptake may be less energetically expensive for 2009a). NHþ 4 þ the fungus as NO 3 first has to be reduced to NH4 prior to incorporation into amino acids. While these N sources must be important for fungal nutrition, the contribution to plant nutrition is controversial (see Read & Perez-Moreno 2003; Leigh et al. 2009) and may depend on the relative N requirements of plant and fungus. In some systems, such as the acidic organic soils frequent in many tropical areas (Moyersoen et al. 2001), AMF NHþ 4 capture and subsequent transfer to the host plant may be of considerable ecological significance. When mycorrhizal roots have access to patches of organic material in soil the AMF appear not to respond (Hodge 2001, 2003b); hyphal proliferation occurs only when the AMF alone have access to the patch (Joner & Jakobsen 1995; Hodge et al. 2001). One remarkable result in Hodge et al. (2001) was that the fungus grew preferentially into a patch of organic matter rather than towards a new, uncolonised host plant. If the fungus gains all its carbon from the host, this implies that the fungus gets an alternative benefit from the patch greater than
Nutritional ecology of AM fungi
that it can gain from a new carbon source. In fact there is good evidence that AMF obtain a growth benefit from organic matter in soil. Leigh et al. (2009) found increased hyphal growth in the ‘plant’ compartment (i.e. that in which both plant and fungus grew) when the fungus was allowed to explore a second, ‘hyphal’ compartment that contained an organic matter patch. Thus, what seems to have been overlooked in the debate over AM fungal responses to organic matter is the possibility that it represents a major N source for the fungus itself. Indeed, there are no studies that directly address the question of the sources of fungal N under realistic conditions. Those that show transfer of N by the fungus to the plant may be revealing a consequence of over-supply of N to the fungus. The demonstration that the supply of organic N compounds can elicit a transcriptional response in G. intraradices (Cappellazzo et al. 2007) adds weight to this suggestion.
Conclusions An increased acceptance of the independent but interacting roles of plant and fungus in nutrient transfers allows a new and necessary focus on the nutritional needs of the fungus itself. All fungi have a high nitrogen content and hence potentially a high N demand. Indeed because AMF are among the most abundant fungi on earth, their role in global N and P cycles would repay close attention. There is abundant evidence that AMF can acquire N (and presumably also P) from decomposing organic material and transfer it to the plant. The N and P transfer to the plant may, if the model proposed above is correct, be a consequence of the fungal demand for nutrients, with both host plant and fungus evolving transporters to take advantage of localised increases in nutrients. However, the mechanisms by which these fungi actively forage in soil for both N and P remain unclear. What is certain is that we need to pay much more attention to the biology and ecology of the extra-radical phase of these fungi if we are to understand how they operate in soil and the roles that they play in ecosystems.
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