Abstract
Ericoid mycorrhizal (ERM) fungi are a diverse assemblage of symbiotic fungi that features culturable ascomycetes in the Helotiales and Onygenales, but also so far unculturable basidiomycetes in the Sebacinales. They form a distinct endomycorrhizal association with some plant genera in the Ericaceae. ERM plants dominate in heathlands characterised by very poor nutrient status and considerable edaphic stress, and their success in these harsh environments is ascribed to the functional traits of their symbiotic fungi. ERM fungi are able to exploit recalcitrant organic substrates thanks to an arsenal of extracellular enzymes. They also display adaptive mechanisms of stress tolerance and are able to withstand high concentrations of toxic compounds such as heavy metals. ERM plants are also commonly found as understorey vegetation in woodland habitats, and molecular investigations on the genetic diversity of ERM fungi, together with cross-inoculation experiments under gnotobiotic conditions, indicate the potential networking ability of these fungi in mixed plant communities.
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I. General Features of Ericoid Mycorrhiza
The aim of this chapter is to review classical and more recent findings on the taxonomic diversity and functional traits of soil fungi forming ericoid mycorrhiza (ERM) with plants in the family Ericaceae. The cladistic relationships within this plant family have been recently investigated by Kron et al. (2002) using nucleotide sequence data from the nuclear 18S and chloroplast encoded matK and rbcL genes. The phylogenetic analysis indicates that Empetraceae and Epacridaceae, previously considered as separate families, are now placed in tribes within the larger family Ericaceae (Kron et al. 2002). The close relationship between epacrids and ericoid plants is further supported by the finding of phylogenetically close mycorrhizal fungi associated with these plant groups (Chambers et al. 2000; Sharples et al. 2000a).
Plants in the family Ericaceae are widespread in a diverse range of heathland and open forest communities, both in the Northern and in the Southern hemisphere (Read 1991; Sokolovski et al. 2002). Although they are commonly found as understorey vegetation, ericaceous shrubs can become dominant in many heathland habitats found at high altitudes and colder altitudes, as well as in Mediterranean climates. These habitats are characterised by very poor nutrient status and considerable edaphic stress (Cairney and Meharg 2003), and it is thought that the success of Ericaceae in these habitats is due to their endomycorrhizal association. In addition to being more stress tolerant than non-mycorrhizal plants in harsh environments (see Cairney and Meharg 2003), ericoid mycorrhizal plants have been also found to be better competitors in mixed plant communities (Genney et al. 2000; Van der Wal et al. 2009).
A common feature of ericoid plants is the anatomy of their very fine and delicate roots, termed hair roots (Read 1996). Hair roots (Fig. 14.1) have a very narrow diameter, usually less than 100 μm, and consist of an inner stele surrounded by a two-layered cortex and an outermost layer of large epidermal cells. These epidermal cells represent the interface with the soil environment (Smith and Read 2008) and are the only hair root cells to be colonised by ERM fungi. Here, ERM fungi form hyphal coils that usually occupy most of the cell volume and are always surrounded by the invaginated plant plasma membrane (Fig. 14.2; Bonfante-Fasolo and Gianinazzi-Pearson 1979; Peterson and Massicotte 2004). Colonisation of epidermal cells by fungi usually occurs directly from the soil through the outer thickened tangential wall (Fig. 14.1), with limited cell to cell hyphal connections (Massicotte et al. 2005) so that most cells represent individual colonisation units. Epidermal cells are sloughed off in the older parts of the hair roots, leaving exposed the suberised cortex layer. Thus, ERM cells are ephemeral and symbiotic nutrient exchange is likely restricted to the younger parts of the root, where both partners are viable. Unlike other endomycorrhizal associations, such as arbuscular and orchid mycorrhiza, viable ericoid fungal hyphae have been observed in plant cells showing signs of organelle and cytoplasm degeneration (Bonfante-Fasolo and Gianinazzi-Pearson 1979; Duddridge and Read 1982). Mechanisms of nutrient acquisition by the plant that involve digestion of the fungal hyphae, such as tolypophagy, suggested by Rasmussen and Rasmussen (2009) for orchid mycorrhiza, can be therefore excluded.
Colonisation of Calluna vulgaris hair roots by ericoid mycorrhizal fungi. (A) Transverse section of a hair root to show the general structure of the root. Cells of the outer epidermal layer harbour fungal coils formed by a sterile mycorrhizal morphotype. (B, C) Fungal hyphae (fh) entering the epidermal cells of C. vulgaris through the tangential wall. The collapsed cortical cells are visible just underneath the epidermis, surrounding the small central cylinder
Ultrastructure of an epidermal cell of C. vulgaris colonised by a typical fungal coil. (A) The fungal hyphae form a dense coil, which occupies a central position in the cell. (B) Detail of an intracellular hypha showing some fungal compartments (vacuole, mithochondrion) and glycogen deposits. The plant plasma membrane surrounds the hypha. (C) A fungal septum labelled with a gold-labelled cellobiohydrolase shows the presence of beta 1,4 glucans (Courtesy of P. Bonfante)
II. Genetic Diversity of Ericoid Mycorrhizal Fungi
To understand the evolution and ecological role of mycorrhizal symbioses, one of the first steps is to unveil the taxonomic position and functional features of the fungal symbionts. The study of ERM is a good example of how the development of molecular tools to investigate fungal diversity has impacted on our view of the specificity of this symbiosis, considered for long time a highly specific interaction restricted to few taxa of plants and fungi (Straker 1996). It has also modified considerably our understanding of ericoid mycorrhizal functioning in ecosystems, revealing potential hyphal networks previously unsuspected (Chambers et al. 1999; Vrålstad 2004; Grelet et al. 2010).
The first attempts to identify ERM fungi were based on the isolation in culture of endosymbiotic fungi from mycorrhizal roots. Several slow growing sterile mycelia, grouped primarily on the basis of cultural morphology and/or ITS-RFLP profiles, have been isolated worldwide from ericoid plants, and many could re-establish ericoid mycorrhiza when inoculated onto axenic plants in gnotobiotic conditions (e.g. Hutton et al. 1994; Perotto et al. 1996; Hambleton and Currah 1997; Liu et al. 1998; McLean et al. 1999; Monreal et al. 1999; Bergero et al. 2000; Cairney et al. 2000; Chambers et al. 2000; Sharples et al. 2000a; Berch et al. 2002; Bougoure and Cairney 2005a, b).
A. Taxonomic Diversity of Ericoid Mycorrhizal Fungi
The first ERM fungus that could be identified taxonomically was a dark isolate obtained from Calluna vulgaris roots by Pearson and Read (1973a) that was eventually induced to form fruiting bodies in pure culture. This isolate formed apothecia and was classified in the genus Pezizella as P. ericae (Read 1974).
The taxonomic position of P. ericae has since been revised: the new name Hymenoscyphus ericae was proposed by Kernan and Finocchio (1983), but a phylogenetic revision of the Hymenoscyphus genus by Zhang and Zhuang (2004) showed that H. ericae was outside this genus, and the new name Rhyzoscyphus ericae was thus proposed. For several years R. ericae, with its anamorph Scytalidium vaccinii (Dalpé et al. 1989; Egger and Sigler 1993), was the only identified fungal symbiont of Ericaceae. The ERM was thus described as a highly specific association that at that time contrasted with the apparent lack of specificity of other endomycorrhizae, such as arbuscular mycorrhiza.
Most slow-growing mycelia isolated from ERM remained unidentified until molecular tools were developed to investigate their phylogenetic affinities. It turned out that a high proportion of these slow-growing isolates, together with fungi isolated from plants with different mycorrhizal status, formed an aggregate of closely related species in the order Helotiales. This assemblage, named “R. ericae aggregate” by Vrålstad et al. (2000, 2002a) because it included R. ericae, contained four main clades. The clades were further refined by Hambleton and Sigler (2005), who also proposed three new species in the anamorphic genus Meliniomyces as members of the “R. ericae aggregate” (Fig. 14.3). Clade 3 contains most of the proven ERM isolates, including type cultures of R. ericae and S. vaccinii. Clade 1 (Meliniomyces variabilis) contains sequences of ericoid root-associated fungal isolates, previously described as “variable white taxon” (Hambleton and Currah 1997), as well as sequences of root endophytes from other host plants from cold-temperate soils of the Northern hemisphere. Some of these fungi can colonise the roots of both ERM and ECM hosts, but although they formed typical ERM structures in hair root hosts, no true mantle was observed in ECM hosts (Piercey et al. 2002; Vohník et al. 2007). Clade 2 (Meliniomyces vraolstadiae) is a small clade containing sequences from fungi so far only found to form ECM, or to be non-mycorrhizal (Vrålstad et al. 2002b).
(A) One of 5,000 MPTs from an aborted parsimony analysis of the ITS-B data matrix. Sequences AJ534704 and AY394885 were deposited under the name “Cadophora finlandica” (as “C. finlandia”) based on ITS sequence similarity scores. Arrowheads indicate additional species that could be described if Fig. 14.3. (continued) isolates are available for examination. All ingroup sequences were derived from cultures isolated from roots, except for four (♦) derived from DNA extracted directly from roots. A majority of ingroup taxa were resolved into four clades, indicated by stars adjacent to the relevant nodes, corresponding to Meliniomyces variabilis, Meliniomyces vraolstadiae, Cadophora finlandica/Meliniomyces bicolor, and Rhizoscyphus ericae. (B) Strict consensus of all 5,000 MPTs with taxon names removed. Stars indicate the same four clades as in (A) (From Hambleton and Sigler (2005), with permission)
Other ERM fungi have been isolated in culture and were identified as ascomycetes in the genus Oidiodendron (Dalpé 1986, 1989). Resynthesis experiments (e.g. Couture et al. 1983; Dalpé 1986) have demonstrated the ERM nature of Oidiodendron, and their occurrence as ERM symbionts has been confirmed in several Ericaceae (Read 1996; Straker 1996; Xiao and Berch 1996; Allen et al. 2003; Bougoure and Cairney 2005a, b). A comparison of rDNA ITS region sequences suggests that many Oidiodendron isolates may have been misidentified in older studies, and that only O. maius, or phylogenetically close species in the Onygenales, forms mycorrhizal associations with Ericaceae in the field (Hambleton et al. 1998; Lacourt et al. 2002).
Ericoid fungi also encompass so far unculturable members of the Basidiomycota that could be precisely identified only by molecular phylogeny. Early ultrastructural studies provided evidence of basidiomycetes forming ERM in roots of Rhododendron, Calluna and Vaccinium (Bonfante-Fasolo 1980; Englander and Hull 1980; Peterson et al. 1980). PCR amplification of the fungal rDNA from mycorrhizal roots of Gaultheria shallon (Berch et al. 2002; Allen et al. 2003) for the first time revealed sequences of Sebacinales that corresponded to more than half of the cloned ITS sequences. Selosse et al. (2007) recently investigated, by direct PCR amplification of ERM samples and ultrastructural observations, the occurrence of Sebacinales in the Ericaceae and found it to be a common symbiont. All sequences of Sebacinales recovered from ERM plants, together with sequences derived from the newly described cavendishioid mycorrhizal type (Setaro et al. 2006) belonged to clade B identified by Weiss (2004) in the phylogeny of Sebacina.
The frequent finding of unculturable fungi in ERM roots is in contrast with the general observation that ericoid symbionts have good saprotrophic abilities and are able to grow on common culture media (Leake and Read 1991). The use of PCR techniques to amplify fungal DNA directly from mycorrhizal roots has made it possible to identify these unculturable fungi, although their role as mutualistic symbionts in the mycorrhizal association remains unclear.
B. Multiple Fungal Occupancy in Ericoid Mycorrhizal Roots
The morphology of mycorrhizal roots in the Ericaceae indicates that epidermal root cells could potentially function as separate units, challenged and colonised by a variety of fungi resident in the rhizosphere. ITS-RFLP analyses of ericoid fungal isolates from different mycorrhizal plant species has demonstrated that multiple occupancy is a common phenomenon in ericaceous roots (e.g. Perotto et al. 1996; Hambleton and Currah 1997; Monreal et al. 1999; Bergero et al. 2000; Chambers et al. 2000; Midgley et al. 2004c; Bougoure and Cairney 2005a, b). This is revealed by the simultaneous presence of fungi with different ITS-RFLP profiles in the same root system. In addition, more sensitive fingerprinting methods (RAPD and ISS-PCR) showed that distinct genotypes of ERM fungi sharing the same ITS-RFLP type can occur in individuals of C. vulgaris (Perotto et al. 1996) and Epacris pulchella (Curlevski et al. 2009). These data indicate that the roots of ericaceous plants are a complex mosaic where different populations of mycorrhizal fungi coexist, each represented by a variable number of genotypes.
The simultaneous association with many and diverse symbiotic fungi may represent an important strategy to broaden the array of functions in the colonisation of difficult substrates. This hypothesis would require that genetic diversity was mirrored by functional diversity, and that different species of ERM fungi were able to perform different physiological functions. This has been demonstrated for some ERM fungi. For example, as discussed in Sect. III.B, ERM can hydrolyse complex substrates, and they do so to a different extent (e.g. Cairney and Burke 1998; Leake and Read 1991; Varma and Bonfante 1994; Midgley et al. 2004a). Even fungal strains within the same species can perform differently. For example, Cairney et al. (2000) found that isolates of R. ericae display different abilities to utilise inorganic and organic nitrogen sources, and Grelet et al. (2009b) demonstrated that functional differences in N transfer to the host plant shoot are maintained among closely related ERM fungi in symbiosis with their hosts.
Better knowledge of the true ERM diversity in ericaceous plants has changed our perception on the specificity in this association, with a wider number of fungal taxa being involved than once thought, mainly in the ascomycetes but also in the basidiomycetes. However, although this wider number and taxonomic position of potential ERM partners, there is evidence of specificity in the composition of the ERM fungal communities associated with co-existing plant species and different environments.
Bougoure et al. (2007) have investigated the diversity of fungi associated with plants of C. vulgaris and Vaccinium myrtillus along a heathland—forest gradient in Scotland. The assemblages of fungi associated with the two plant species were different, even though plants were co-occurring in the forest understorey. In addition, the community of fungi associated with C. vulgaris hair roots was different for samples collected from the forest, open heathland and a transition zone between the two (Bougoure et al. 2007). Part of the differences were due to the amplification, in the forest samples, of typical ECM ascomycetes and basidiomycetes sequences.
In a more homogeneous environment such as the subarctic heaths, however, Kjøller et al. (2010) investigated the fungal communities associated with the roots of four co-existing ericaceous plants (Empetrum hermaphroditum, Andromeda polifolia, Vaccinium uliginosum, V. vitis-idaea) and could not find significant differences in relationship with the host plants, whereas significant differences in spatial distributions were observed. Thus, it seems that, similarly to what happens in other endo- and ectomycorrhizal symbioses, environmental factors as well as the host plant species may influence, at least in some cases, the ERM fungal community even when a broad range of potential symbionts are available.
C. Potential Networking Abilities of Ericoid Mycorrhizal Fungi
The phylogenetic analyses described in the previous paragraphs demonstrate that a diverse assemblage of fungi can interact with Ericaceae to form ERM. Conversely, the occurrence of ERM-forming fungi on plant species outside the Ericaceae has also been frequently demonstrated. Duckett and Read (1995) reported that R. ericae, the best studied ERM symbiont, was also capable of forming in vitro intracellular coils in the rhizoids of liverworts.
Further support to this observation derived from molecular studies on leafy liverworts in the genus Cephaloziella. Mycelia isolated in nature from liverworts and demonstrated to form ERM on ericaceous hosts were first assigned to the genus Hymenoscyphus (syn. Rhizoscyphus) by Chambers et al. (1999). Later on, Upson et al. (2007) performed Koch’s postulate for the C. varians—R. ericae association, inoculating axenically grown liverwort with an isolate of the fungus from the plant. These isolates from Antarctica could also colonise Vaccinium macrocarpon roots, despite the absence of ericaceous plant species from maritime and continental Antarctica.
ERM and ECM plants often co-exist in natural ecosystems such as boreal or Mediterranean forests, where ericaceous plants constitute the understorey vegetation of dominant ECM tree species (Read 1991). The traditional view that ECM and ERM fungi are taxonomically distinct has been challenged by several papers reporting amplification of ERM sequences from ECM roots. An important discovery by Vrålstad et al. (2000) was the strong genetic similarity among fungi of the R. ericae aggregate associated with ERM and ECM roots in a boreal forest.
Following ITS sequence comparison, the ERM fungus R. ericae was the closest relative of fungi isolated from Piceirhiza bicolorata, an ECM morphotype common in post-fire sites. Phylogenetic analyses showed that fungi from ERM and ECM roots, as well as other endophytes, belong to different clades of the “R. ericae aggregate”, first proposed by Vrålstad et al. (2002a) and later revised by Hambleton and Sigler (2005).
None of the fungal isolates from P. bicolorata and grouped in Clade 1 of the R. ericae aggregate (Fig. 14.3), corresponding to M. variabilis, could form ERM on ericaceous hosts (Vrålstad et al. 2002b), although a M. variabilis isolate from P. bicolorata ECM has been recently found to form ERM in V. vitis-ideae (Grelet et al. 2010).
Isolates assigned to the C. finlandica/M. bicolor (Clade 4 in Fig. 14.3) have been found to form ECM or ectendomycorrhiza with Betula, Picea and Pinus (Wilcox and Wang 1987; Vrålstad et al. 2002b). An isolate of M. bicolor derived from Piceirhiza bicolorata ECM was also demonstrated to form, at the same time, ERM on V. myrtillus and ECM with Pinus sylvestris, and to induce beneficial effects on host plant growth (Villarreal-Ruiz et al. 2004). This finding confirms previous observations by Monreal et al. (1999) that Phialophora (= Cadophora) finlandica could form, at least in vitro, ERM with Gaultheria shallon. Additional observations derive from the work of Grelet et al. (2009a), showing that M. bicolor obtained from Piceirhiza bicolorata ECM formed typical ERM structures and engaged in reciprocal transfer of carbon and nitrogen with V. vitis-idaea.
Bergero et al. (2000) demonstrated that several fungi (including Oidiodendron spp. and sterile morphotypes) in a Mediterranean forest were associated with both ERM and ECM plants. Fungi isolated from root tips of ECM Quercus ilex formed ERM with Erica arborea in vitro, and molecular analyses indicated that some of these Q. ilex fungal associates were conspecific with ERM fungi naturally occurring in E. arborea roots.
Potential Hyphal Connection Between ERM and Non-Ericaceous Plants Have Been Further Identified
For example, at least six ITS-RFLP types of ERM fungi, including Helotiales and Oidiodendron, were common root associates of a diverse array of plant taxa within a schlerophyll forest community in south-eastern Australia (Chambers et al. 2008). Similar results were obtained by Curlevski et al. (2009) and Grelet et al. (2010), who applied the more sensitive inter-simple sequence repeat (ISSR)-PCR to investigate the occurrence of fungi with the same ISSR fingerprints in co-existing ERM and ECM plants. Shared fungal genotypes were identified between the ERM species E. pulchella and the ECM species Leptospermum polygalifolium (Curlevski et al. 2009) in a schlerophyll Australian forest, and between the ERM species V. vitis-idaea and the ECM species P. sylvestris (Grelet et al. 2010) in a Scottish boreal forest.
From the experiments described above, there is now strong evidence that potential hyphal connections between ERM and ECM plants are a common feature in different forest ecosystems, although the work by Grelet et al. (2010) would exclude the formation of large mycelial networks at least for M. variabilis, given the small genet size found for this fungus (<13 cm). However, several questions remain open on the type of interactions that ERM fungi may establish with non-ericaceous host plants, and on the functional role of these potential hyphal connections.
Some observations suggest that, rather than forming an ECM association themselves, many ERM fungi may associate with ECM root tips in addition to the true ECM partner.
Although one isolate of the C. finlandica/M. bicolor clade in the “R. ericae aggregate” has been demonstrated to form both ERM and ECM, or ectendomycorrhizal, associations with tree species under gnotobiotic conditions (Villarreal-Ruiz et al. 2004), most other isolates seem to be able to form a single mycorrhizal type, despite their common occurrence in plant roots. Bergero et al. (2000) clearly showed, in their inoculation experiments, that ERM-forming fungi isolated from ECM Q. ilex were often morphologically distinct from those producing the ECM. Similarly, there is no clear evidence that M. variabilis can actually form ECM, and it seems more likely that M. variabilis is an endophyte in ECM root tips formed by other fungi (Hambleton and Sigler 2005; Grelet et al. 2010). This would give support to the observation of endophytic behaviour of M. variabilis (Vohník et al. 2007; Ohtaka and Narisawa 2008), or the amplification of Helotiales DNA sequences from ECM formed by other fungal species (Tedersoo et al. 2009).
Occurrence of ERM Ascomycetes in Non-Ericaceous Hosts Is Not Restricted to ECM-Forming Plants
As already mentioned, Helotiales forming ERM coils in hair roots of Woollsia pungens (Ericaceae) under gnotobiotic conditions were isolated from the roots of 17 species from different plant families (Apiaceae, Cunoniaceae, Cyperaceae, Droseraceae, Fabaceae—Mimosoideae, Lomandraceae, Myrtaceae, Pittosporaceae, Proteaceae, Stylidiaceae) at an Australian sclerophyll forest site (Chambers et al. 2008).
In cold climates, dark septate endophytes (DSE) are widely distributed and frequently isolated from the roots of several plant species (see references in Jumpponen and Trappe 1998; Mandyam and Jumpponen 2005). Some members of the “R. ericae aggregate” are well recognised DSE: in addition to M. variabilis, Cadophora finlandica is commonly identified in the roots of mainly ECM plant species (Wilcox and Wang 1987; Mandyam and Jumpponen 2005). They have been identified also in Antarctica on Colobanthus quitensis and Deschampsia antarctica (Newsham et al. 2009). The ecological functions of DSE are not well understood, but they can provide several benefits to their host plants, including facilitation of nutrient uptake, protection from metal stress tolerance and stimulation of the mycorrhizosphere community against root diseases (Jumpponen 2001; Mandyam and Jumpponen 2005; Dos Santos Utmazian et al. 2007; Alberton et al. 2009).
A study by Abuzinadah and Read (1989) showed that Oidiodendron also enhances growth of Betula pendula on a medium containing proteins as sole nitrogen source, without producing a classical ectomycorrhizal infection. Similarly, endophytic fungi isolated from roots of the grass Deschampsia flexuosa and identified in the Helotiales have been shown to colonise roots and enhance nitrogen uptake by C. vulgaris (Ericaceae) seedlings, and vice versa (Zijlstra et al. 2005). As discussed by Curlevski et al. (2009), many questions remain on the relationships between fungi that form ERM and DSE associations, particularly with regard to the benefits conferred to plants by the DSE infection (Schulz and Boyle 2005).
The ability to infect multiple hosts raises the possibility that ERM associations in ericoid roots and DSE associations in non-ericaceous roots might represent parts of a common mycelial network.
Hyphal links playing important functions in nutrient exchange, including organic carbon, have been demonstrated for ECM fungi (Simard et al. 1997), and for more complex situations involving plants with different mycorrhizal status (e.g. McKendrick et al. 2000; Bidartondo et al. 2003). The role of possible physical links between ericaceous and non-ericaceous plants via their fungal associates remains an open question, as well as the nature of the functional relationships with these hosts. It also remains to be established whether or not the same fungus can form different types of mycorrhizal symbioses with distinct hosts in natural conditions. The classical bioassay to establish the mycorrhizal nature of a fungal isolate is the inoculation in vitro onto axenic seedlings. Although this assay remains an important test to elucidate the mycorrhizal potential of a fungal isolate, the conditions used are very different from those found in nature and the results must be interpreted with caution (Read 1996).
In addition to a possible role in nutrient exchange, the association of ERM fungi with non-ericaceous plants may be ecologically relevant in some stressed conditions. For example, genetic relatedness between ERM and ECM fungal associates was found in forests subjected to fire (Bergero et al. 2000; Vrålstad et al. 2000), and it was suggested that the ECM host, with deeper roots, could provide an efficient source for biotrophic infection of the ERM (or other) plants. A role as a reservoir of ericoid fungi has also been suggested for liverwort rhizoids by Duckett and Read (1995).
III. Exploitation of Inorganic and Organic Substrates by Ericoid Mycorrhizal Fungi
Habitats dominated by Ericaceae include most humus heathlands in the Northern hemisphere, Mediterranean woodlands, tropical cloud forests and the dry sand plains of Australia (Read 1991; Straker 1996; Bergero et al. 2000). Soils colonised by ERM plants are characterised by the low availability of nutrients, due to slow litter decomposition and mineralisation processes. Here, essential nutrients such as N and P are found almost exclusively in organic forms (Read and Perez-Moreno 2003). In these ecosystems, the dominant plant species are highly dependent on the mycorrhizal symbionts for their nutrient supply. Although ERM fungi appear to have a poorly developed extraradical mycelial phase, they are able to mobilise organic nutrients rendering them accessible to the host roots (Finlay 2008).
In mor-humus heathlands, the limiting nutrient is primarily nitrogen, which is in the form of acid-hydrolysable organic compounds and insoluble humin, usually inaccessible to plants (Stribley and Read 1974). The success of Ericaceae in these stressful habitats is therefore largely related to the abilities of their ERM symbionts to improve nutrient acquisition, particularly nitrogen, from organic forms. Several studies on ERM mycelia growing in vitro (see Smith and Read 1997, 2008) have confirmed their high saprotrophic capabilities, which would facilitate nutrient mobilisation from the substrates exploited by the mycorrhizal roots and the mycelium. For example, Read and Perez-Moreno (2003) pointed out that the enzymatic capabilities of ERM fungi could extend the sources of N important to their plant hosts beyond inorganic forms to include amino acids, amino sugars, proteins, or chitin. In return for N and other nutrients, plants probably allocate up to 20 % of net primary production to their mycorrhizal symbionts (Hobbie 2006). Mobilised nutrients from organic forms can then be assimilated by plant roots, either directly or via the fungal intermediary.
A. Nitrogen and Phosphorus Uptake
Organic nitrogen (ON) accounts for up to 95 % of the soluble N pool in soils (Abuarghub and Read 1988; Talbot and Treseder 2010). This pool represents an important component of plant N budgets, considering that inorganic N pools in soil are insufficient to account for annual plant N uptake in many ecosystems (Kielland 1994). Mycorrhizal plants have greater access to ON than non-mycorrhizal plants (Schimel and Bennett 2004). The uptake of ON by ERM fungi and the subsequent transfer to the plant require multiple steps, including breakdown of polymers in soil solution, direct uptake of mono- and oligomer into mycorrhizal fungi, internal transformation of ON, and transfer across the fungus—host plant interface (Talbot and Treseder 2010). Identifying which mechanisms most strongly control each of these steps will help in understanding the extent of ON usage by mycorrhizal plants (Talbot and Treseder 2010). The major classes of ON compounds found in soils and in soil solution include aliphatic-N, like amino-N and polysaccharide-N, and aromatic-N, such as the compounds present in soil humus (Roberts and Jones 2008). The high soil ON content indicates that the exposure of mycorrhizal roots to ON can be equal to or greater than exposure to inorganic N in most soils (Talbot and Treseder 2010). The forms of ON that are transferred to the plant may vary among fungal and plant species, with glutamine, asparagine, and alanine being the most common ON compounds involved in this process (Chalot and Brun 1998). Systems that are dominated by ERM fungi that have the capability to produce a broad range of extracellular enzymes, such as R. ericae (Cairney et al. 2000; Midgley et al. 2006), may present a particularly high rate of ON uptake.
A significant pool of available nitrogen in heathland soils is represented by free amino acids (Abuarghub and Read 1988). R. ericae has been shown to absorb a broad range of amino acids (Chen et al. 1999; Whittaker and Cairney 2001) and to effect transfer of aminonitrogen to its hosts, although considerable intraspecific variation can exists in this regard (Bajwa and Read 1986; Cairney et al. 2000). The utilisation of sulfur-containing amino acids by R. ericae is poor but, in common with other filamentous fungi, this may be enhanced under sulfur starvation (Bajwa and Read 1986). In vitro culture studies indicate that some ERM fungi can use amino acids as their sole N source (Talbot and Treseder 2010). Amino acids vary in their frequency within proteins, with leucine, glycine and alanine being most common and tryptophan, cysteine and histidine rarest. Talbot and Treseder (2010) found that the percentage of ERM fungal species capable of using a given amino acid was significantly and positively correlated with its relative abundance in proteins.
Early work on the physiology of ERM fungi and their influence on plant nutrient uptake was on a limited number of ERM strains, mainly R. ericae (Leake and Read 1991). However, a single root system can harbour several ERM fungal genotypes, which may also belong to different species. It became therefore important to understand whether this genetic diversity confers functional diversity which could be advantageous in nutrient-poor habitats.
Cairney et al. (2000) studied various fungal strains isolated from hair roots of C. vulgaris and showed significant variations in the N utilisation pattern. While most isolates showed a preference for ammonium as a sole nitrogen source, considerable variation was observed in the abilities of isolates to utilise amino acids and proteins (e.g. BSA). In particular, large intraspecific variation was observed in the use of glutamine and BSA. Individual isolates of R. ericae may therefore vary considerably in their abilities to use organic nitrogen from different substrates in soil. Grelet et al. (2005) showed that the different abilities to utilise organic and mineral N sources were affected by carbon availability in a strain-specific manner. Under elevated C supply, growth differences among strains were linked to the total amount of nitrogen taken up, suggesting variation in uptake kinetics. But, under C-limiting conditions, the nitrogen-use efficiency explained strain differences, implying intraspecific variations in N metabolism. The main effect of reducing C availability was to increase the relative ability of most strains to grow on glutamine and nitrate, in comparison to ammonium. Grelet et al. (2009b) investigated whether the functional variation was maintained in symbiosis. Vaccinium plants were inoculated with three genetically closely related ERM fungal strains known to differ in their N use in liquid culture. 15 N was used to trace N uptake into shoots, and the results demonstrate that functional differences among closely related ERM fungi are maintained in symbiosis with their hosts, thus suggesting that N transfer to plant shoots in ericoid mycorrhiza is under fungal control. In the experimental conditions used by Grelet et al. (2009b), the strain-specific effect was evident when N was supplied as ammonium or glutamine, but fungal influence on plant N uptake has also been shown for a range of amino acids (Sokolovski et al. 2002) and for nitrate (Kosola et al. 2007).
These experiments give support to the hypothesis that a broad spectrum of symbiotic ERM fungi may provide the host plant with a wider set of acquired symbiotic functions.
In acidic heathland soils, a very low amount of free inorganic phosphate is found, and the main phosphorus sources are organic compounds in the form of phosphomonoesters, mainly phytates (Cosgrove 1967; Mitchell and Gibson 2006), or phosphodiesters such as nucleic acids (Griffiths and Caldwell 1992). Phytates are usually complexed with iron and aluminium, and ERM fungi are able to access these sources (Mitchell and Read 1981). Both extracellular and wall-bound phosphatases are produced by a number of ERM isolates (Straker and Mitchell 1986). Phosphomonoesters are quantitatively the most important fraction of organic soil phosphorus, while phosphodiesters are found in relatively low concentrations but are potentially valuable phosphorus sources (Griffiths and Caldwell 1992; Leake and Miles 1996; Myers and Leake 1996). Leake and Miles (1996) demonstrated the ability of R. ericae to degrade DNA molecules through the production of phosphodiesterases and to directly assimilate nucleotides.
Pearson and Read (1973b) provided evidence for a movement of P-32 orthophosphate from the fungus to the plant. Two phosphate membranes transport systems were identified for a South African isolate as high affinity and low affinity systems (Straker and Mitchell 1987). Interestingly, both P uptake systems operate better under the pH conditions typically found in the acidic soils where ERM fungi live.
Many fungi can dissolve phosphate and other essential nutrients from mineral substrates by employing several mechanisms, including protonation and chelation (Gadd 2010). In terms of mineral weathering and dissolution, mycorrhizal fungi form one of the most prominent groups of soil microorganisms (Devevre et al. 1996; Jongmans et al. 1997; Lundström et al. 2000). A number of ERM fungi has been investigated for their phosphate solubilising ability. Van Leerdam et al. (2001) showed that most isolates could solubilise the rock phosphate hydroxyapatite in the presence of ammonium, whereas Martino et al. (2003) and Gibson and Mitchell (2004) showed that O. maius and R. ericae-type isolates were capable of solubilising zinc phosphate, and some of these isolates were also able to solubilise calcium phosphate.
B. Depolymerisation of Complex Substrates
The array of hydrolytic enzymes produced by ERM fungi enables their saprotrophic growth in the absence of the host (Perotto et al. 1993, 1997), but may also mediate utilisation of organic nutrient sources by the host plant (Cairney and Burke 1998). Ericoid fungi produce a range of extracellular enzymes that catalyse breakdown of several organic macromolecules and give plants access to breakdown products of complex polymers that cannot be directly assimilated by either plant roots (Read and Perez-Moreno 2003). This access appears particularly important in N-limited systems, where mineralisation is insufficient to support plant N demand (Schimel and Bennett 2004).
Two classes of polymer-degrading enzymes can be recognised (Read and Perez-Moreno 2003). The first class includes several hydrolases that cleave the nutrient containing molecules themselves, whereas the second class facilitates nutrient acquisition by attacking organic molecules such as lignins, polyphenols and tannins, which may protect or precipitate essential nutrients. Lignases and polyphenol oxidases are comprised in this type of enzymes, and are expected to give a major contribution to litter decomposition as well as to plant nutrition.
ERM fungi express both types of polymer degrading enzymes. In addition to proteases and chitinases, they produce hydrolytic enzymes, which degrade cell wall polysaccharides such as cellulose, hemicelluloses and pectin, as well as phenol oxidase activities, which may facilitate host access to nitrogen and phosphorus within moribund plant material or from polyphenol complexes in soil (Perotto et al. 1993, 1997; Varma and Bonfante 1994; Bending and Read 1996a, b; Burke and Cairney 1997a, b; Cairney and Burke 1998; Piercey et al. 2002; Midgley et al. 2004a, 2006).
The complete use of cell wall polysaccharides, such as cellulose, is not surprising, as these enzymes are presumably required for host cell penetration. Like cellulose, the potential of ERM fungi to degrade pectin is important during penetration of the root of the host. ERM fungi have been shown to produce polygalacturonases in culture (Perotto et al. 1993, 1997; Midgley et al. 2006). R. ericae is also able to grow on xylans thanks to the production of at least one xylanase and a range of accessory enzymes (Burke and Cairney 1997a, b).
Polyphenol oxidases are known to be produced by ERM fungi (Burke and Cairney 2002). These enzymes include laccase, catechol oxidase and tyrosinase, which show a considerable overlap in substrate affinities (Burke and Cairney 2002). R. ericae produces laccase, along with a range of related phenol-oxidising activities (Burke and Cairney 2002). It has been suggested that laccases produced by ERM fungi may be engaged in a number of processes involved in the functioning of the symbioses. Suggested roles include lignin and polyphenol degradation, release of N from insoluble protein—tannin complexes and degradation of polycyclic aromatic hydrocarbon pollutants (Leake and Read 1989; Hutchison 1990; Bending and Read 1996a, b, 1997; Braun-Lüllemann et al. 1999). Cairney and Burke (1998) assessed the production of ligninolytic activities by R. ericae. The fact that R. ericae releases hydrogen peroxide (Bending and Read 1997) and hydroxyl radicals (Burke and Cairney 1998) into culture media confirms that the fungus may contribute to a form of lignin degradation, similar to that seen in “brown rot” fungi, in which the fragmentation of the lignin polymer is mediated by these radicals (Burke and Cairney 1998; Cairney and Burke 1998). Carbohydrate oxidase activity may further release H2O2, which, via production of hydroxyl radicals in the presence of Fe, may contribute to partial lignin degradation by this fungus. Burke and Cairney (1998) examined isolates of the ERM fungus R. ericae for their ability to oxidize carbohydrates to their corresponding lactones and to excrete the H2O2 produced thereby. R. ericae was found to express cellobiose oxidase and glucose oxidase when grown on cellobiose and glucose respectively.
Polyphenolic compounds can reach high concentrations in heathland soils (Jalal and Read 1983). In these phenol-rich environments, the proven ability of ERM fungi to use many monomeric phenolic compounds as carbon sources (Leake and Read 1989) and to release the enzymes laccase and catechol oxidase (Bending and Read 1996a, b, 1997), involved in the degradation of hydrolysable polyphenols, plays a fundamental role in plant nutrition.
Bending and Read (1996b) demonstrated that, in addition to degrading tannic acid-protein precipitates by releasing polyphenol oxidase, R. ericae and Oidiodendron sp. were able to acquire nitrogen from these complexes through the expression of extracellular acid proteases. Such observations indicate that the ability to absorb amino acids bypassing the N mineralisation process may be widespread in organic soils. R. ericae is in fact known to produce extracellular proteinases that, via the hydrolysis of simple proteins, release amino acids that can be absorbed by the mycelium (Read et al. 1989; Whittaker and Cairney 2001). Interestingly, R. ericae also secretes an extracellular proteinase identified as a carboxyl (acid) proteinase (Leake and Read 1989) that showed optimal activity at pH 2.2, which is considerably lower than that found in acidic heathland soils (Leake and Read 1990b). However, most of the fine mycorrhizal roots of ericaceous plants are confined to the soil/litter interface, where, due to the activity of H+-releasing processes, the pH is lower than in the bulk soil (Read et al. 1989). This interface is also the location where protein substrates accumulate. The proteinase activity of the fungus is, therefore, adapted to the specific micro-environment in which it is found. Moreover, as the pH within plant cells approaches neutral, the low pH optimum of the proteinase may also protect the plant from protein degradation (Leake and Read 1989). It has also been proposed that excess proteinase is only released by the fungus if a suitable substrate is present in the soil (Leake and Read 1990c). The end products or amino acids can be taken up directly by the ERM fungus without further deamination and these forms of nitrogen would also be ideal for transfer from the fungus to the host plant.
ERM fungi from Ericaceae growing in the Southern hemisphere appear to be broadly similar to R. ericae in their abilities to utilise different carbohydrates sources, as well as amino acids and proteins as nitrogen sources (Chen et al. 1999; Whittaker and Cairney 2001; Midgley et al. 2004b).
Using crustacean chitin as a model compound, it was demonstrated that R. ericae and Oidiodendron griseum could cleave the polymer to its constituent subunits, which were readily assimilated (Leake and Read 1990a; Mitchell et al. 1992). A significant proportion of the N acquired by the fungus was found to be transferred to aseptically grown mycorrhizal plants of V. macrocarpon (Kerley and Read 1995). R. ericae has also been shown to supply its host plant with nitrogen directly from chitin (Kerley and Read 1997), providing strong evidence that chitinase activities are produced by the fungus during symbiosis. Bougoure and Cairney (2006) also showed that an isolate of R. ericae from E. pulchella produced both endo- and exo-acting chitinolytic activities.
Concerning the acquisition of P from polymeric organic sources, both DNA in purified form (Leake and Miles 1996) and entire nuclei (Myers and Leake 1996) have been shown to represent useable P sources for V. macrocarpon. P mobilisation appeared to depend upon production of phosphodiesterases by R. ericae (Leake and Read 1997).
A significant consumption of C and N is required for extracellular enzyme production (Schimel and Weintraub 2003). In fact, it has been shown that plant C supply to the fungal symbiont can control extracellular enzyme production and the acquisition of nutrients from polymeric compounds, and that ERM fungi acquire more N from high molecular weight organic nitrogen compounds when grown with the plant (Bajwa and Read 1986; Dighton et al. 1987; Gunther et al. 1998). Under elevated glucose concentrations in the culture medium, R. ericae acquired a higher percentage of N from glutamine (Grelet et al. 2005), and protein mineralisation by other ERM fungal isolates also increased (Zhu et al. 1994; Eaton and Ayres 2002). Under the same glucose concentration, the expression of extracellular proteases (Nehls et al. 1999) and chitinases (Leake and Read 1990a; Bougoure and Cairney 2006) decreased in other ERM fungi. There is therefore evidence that C availability increases uptake of amino acids by ERM fungi, and that a strong C regulation of extracellular enzyme activities operates by induction/repression mechanisms (Talbot and Treseder 2010).
IV. Ericoid Mycorrhizal Fungi in Soils Enriched in Toxic Compounds
Since the first investigations by Bradley et al. (1981, 1982), the success of Ericaceae in stressful habitats has been ascribed to the unique abilities of their ERM fungal partners to withstand and adapt to environmental stresses and to enhance stress tolerance in their host plants (Cairney and Meharg 2003).
Environmental stress can shape adaptation and evolution of organisms living in changing environments, and the impact of human activities on natural environments has caused rapid and often stressful and deteriorating changes. The occurrence of adaptive processes is clearly shown by the fact that many organisms have acquired quite rapidly the ability to withstand man-made changes in the environment (Nikolaou et al. 2009).
Heavy metal toxicity represents a strong selection pressure and microbial resistance to toxic metals is widespread, with frequencies ranging from a few per cent in pristine environments to nearly 100% for bacteria growing in heavily polluted environments (Silver and Phung 2009). Adaptation of ecto- and endomycorrhizal fungi to heavy metal soil pollution of anthropic origin is also supported by several studies (e.g. Leyval et al. 1997; Meharg and Cairney 2000; Colpaert et al. 2004; Adriaensen et al. 2005; Krznaric et al. 2009).
A. Genetic and Functional Adaptation to Heavy Metals
As already described, soils naturally colonised by Ericaceae are generally acidic. Low pH and anaerobic soil conditions facilitate mobilisation of heavy metal ions, which are toxic above threshold concentrations (Meharg and Cairney 2000). Bradley et al. (1981, 1982) first demonstrated the importance of ERM fungi to increase resistance of C. vulgaris to heavy metals, and other authors later described metal tolerance in fungal isolates from sites with different pollution. In particular, ERM plants in association with members of the “R. ericae aggregate” have been shown to survive in arsenate-rich sites (Sharples et al. 2000b) and populations of arsenate resistant R. ericae have been isolated from C. vulgaris growing in As/Cu-contaminated mine soils (Sharples et al. 2001). A detailed study on more than 70 ERM fungi from polluted and non-polluted sites indicated an adaptive resistance to arsenate in the fungal populations from the mine site (Sharples et al. 2001). Strains of the ERM species O. maius also showed adaptation to metal pollution, as isolates from an industrial site heavily polluted with Cd, Zn and Al displayed better growth in vitro on media containing these metals, when compared with isolates from non-polluted sites (Martino et al. 2000a).
Ericoid fungal strains of O. maius derived from polluted and unpolluted soils also mobilised insoluble inorganic zinc compounds to different extents (Martino et al. 2003). Strains from polluted soils showed little ability to solubilise Zn from both ZnO and Zn3(PO4)2, whereas strains from unpolluted soils showed a higher solubilisation potential. As solubilisation of insoluble metal compounds may lead to toxic metal concentrations in soils enriched with heavy metals, this mechanism may be adaptive.
In addition to being polluted by human activities, soils may be naturally enriched in heavy metals derived from specific rock substrates. Serpentine soils derived from ultramafic rocks are low in plant nutrients such as K and Ca, but contain high levels of potentially toxic elements such as Ni and Cr. Vallino et al. (2011) compared metal tolerance of O. maius isolates derived from sites with contrasting metal pollution (industrial Cd/Zn pollution versus natural Cr/Ni pollution). Despite the small sample size, a significant difference was observed in growth of the O. maius isolates on media containing these metals. Strains more tolerant to Cr and Ni were those originated from the serpentine site, while strains more tolerant to Zn and Cd were isolated from the industrial sites polluted by these contaminants (Vallino et al. 2011). This would suggest, at least for some metals, a specific adaptation of O. maius that reflects the specific contamination in the soil of origin.
For other metals, such as copper, adaptive heavy metal tolerance is more controversial. For example, ERM fungi of C. vulgaris have been shown to display constitutive tolerance to copper (Bradley et al. 1982; Sharples et al. 2001). However, extracellular phosphodiesterase activity was stimulated by copper in ERM fungal isolates from mine spoil sites but not in fungi from uncontaminated sites (Gibson and Mitchell 2005). This result has been interpreted as an adaptive metal avoidance mechanism, as phosphate ions released from organic substrates by phosphatases would react with metal ions to form insoluble, and therefore less toxic, compounds (Gibson and Mitchell 2005).
Increased metal tolerance in ERM fungi can therefore arise, under selective pressure, as a consequence of new or modified phenotypic traits. As the phenotype depends on the cell genetic information, changes in gene sequences and arrangement may be expected to have arisen in those ERM fungi adapted to environmental metal stress. Mutations are one of the primary sources of genetic variation, and many environmental agents are known to induce mutations either directly or indirectly, through the production of reactive oxygen species (Hartwig 1995).
A recent study (Vallino et al. 2011) suggests that the mutation rate positively correlated with environmental stress within the same ERM fungal species, and that mutations do not occur randomly in the genome. Two gene regions with different functional roles in heavy metal resistance (the ribosomal ITS and the gene coding for the Cu/Zn superoxide dismutase) were compared among isolates of O. maius derived from non-polluted soil, and from soils with natural and industrial heavy metal pollution (Vallino et al. 2011).
The ribosomal ITS region can be considered as a “neutral” gene towards metal tolerance, whereas Cu/Zn SOD plays a major role in cell defence against toxic reactive oxygen species, which are increased by environmental stress, including heavy metals (Schützendübel and Polle 2002). The role of the Sod1 gene (coding for the O. maius Cu/Zn SOD) in metal tolerance was recently demonstrated in O. maius by Vallino et al. (2009) and Abbà et al. (2009).
As compared with the “neutral” ITS, a higher mutation rate was found in the functional Sod1 locus of strains from all sites (Fig. 14.4), suggesting that genes with a functional role in fungal survival display a higher nucleotide polymorphism than neutral genes (Vallino et al. 2011). Moreover, a significantly higher mutation rate was found in isolates from heavily polluted industrial soils, when compared with isolates from non-polluted or naturally polluted soils (Fig. 14.4). These observations are in agreement with the finding, derived from genomic comparisons, that fungal stress signalling pathways are evolving rapidly and in a niche-specific fashion to protect different species against the contrasting environmental stresses they encounter in their diverse habitats (Nikolaou et al. 2009). Interestingly, in the O. maius isolates from polluted sites, most mutations were in the Sod1 gene promoter region, rather than in the coding sequence (Vallino et al. 2011), suggesting that mutagenesis induced by environmental stress may also target specific gene regions. A similar situation was observed in the promoter region of metallothionein genes in a cadmium tolerant population of the collembola Orchesella cincta (Janssens et al. 2007).
Sequence polymorphism of the ITS and the SOD loci. (A) p-Distance of the ITS region and the SOD in ericoid mycorrhizal strains derived from three sites respectively non-polluted, naturally polluted (serpentine) and industrially polluted. (B) p-Distance of different regions of SOD locus in the same three groups of fungi (Modified from Vallino et al. (2011))
B. Cellular and Molecular Mechanisms of Heavy Metal Tolerance
Metals are directly or indirectly involved in all aspects of microbial growth, metabolism and differentiation (Gadd 1993, 2010). Several metals play essential functions in the organisms, and insufficient levels of essential metals can result in stress responses just as severe as those resulting from excess metals. Therefore, cells must have developed mechanisms to maintain metal homeostasis (Tomsett 1993). By contrast, some heavy metals such as Cs, Al, Cd, Hg and Pb have no known functions in most organisms and are therefore toxic at all concentrations. Molecular recognition allows organisms to differentiate between essential and non-essential ions and, if necessary, to partition them in different ways.
The cellular and molecular mechanisms potentially involved in metal tolerance in fungi, like in other organisms, can be classified into three groups: (i) avoidance mechanisms that restrict entry of metal ions into the cytoplasm, and relies on decreased uptake or increased efflux of metal ions, or by their immobilisation outside the hypha by biosorption to cell walls, pigments and extracellular polysaccharides, (ii) sequestration mechanisms that reduce the concentration of free metal ions in the cytosol and include metal-binding peptides and proteins which regulate metal ion homeostasis, as well as intracellular compartmentation and (iii) antioxidative mechanisms that allow the fungus to directly or indirectly counteract accumulation of ROS and oxidative stress. Some of these mechanisms are constitutively present, whereas others are only activated when metals exceed a threshold value (Colpaert et al. 2011). Most information on the mechanisms of heavy metal tolerance in mycorrhizal fungi derive from work on ECM fungi (e.g. see Courbot et al. 2004; Bellion et al. 2006; Colpaert et al. 2011), although the most recent work on ERM fungi, described later in this chapter, opens up the possibility to use some ERM isolates as model systems to investigate heavy metal tolerance.
Fungal mycelia often have a high sorption capacity for metals, so binding of metals to the hyphal surface may represent a substantial fraction of the metal accumulated by mycelia (Gadd 1993, 2010; Leyval et al. 1997). Biosorption to cell walls, pigments and extracellular polysaccharides have been reported for ERM fungi (Bradley et al. 1981; Denny and Ridge 1995; Martino et al. 2000a).
Fungal weathering can release essential metals and nutrients from insoluble minerals, but it can also increase final concentrations of toxic metals in the soil. Biotransformation of solubilised metals into insoluble organic forms is a common phenomenon in both ECM and ERM fungi (Gadd 2010). Biotransformation of Zn was observed in O. maius isolates from soils heavily polluted with heavy metals which were found to cause precipitation of organic zinc (Martino et al. 2003). Similarly, R. ericae exposed to uranium demonstrated a high tolerance to uranium oxides and was found to form extracellular crystalline precipitates of uranium-containing minerals on the fungal mycelia (Fomina et al. 2007), likely with the involvement of oxalic acid.
Fungi can respond to the presence of metals with the release of specific proteins in the surrounding medium. It was found for example that the presence of zinc in the culture medium sharply increased in O. maius the secretion and activity of extracellular enzymes that hydrolyse the pectin component of plant cell walls (Martino et al. 2000b). The significance of this increased production is unclear, but oligalacturonans may function as better metal chelators than larger polymers, thus protecting the fungus during saprotrophic growth. Zinc ions also induced in O. maius a general change in the array of secreted proteins, with a shift towards the production of more basic, low molecular weight polypeptides (Martino et al. 2002).
Intracellular metal concentrations in fungi may be regulated by transport, including efflux mechanisms and internal compartmentation. Metal transporters are major players in keeping the cytosolic concentrations compatible with metabolic activities, and they may either extrude metal ions out of the cell as plasma-membrane transporters, or sequestrate metals in intracellular compartments, usually the vacuole (Fig. 14.5). Most of our knowledge on metal transporters in fungi comes from studies on the yeast Saccharomyces cerevisiae (Rutherford and Bird 2004), but both mechanisms have been identified in ECM fungi (e.g. Blaudez et al. 2000; Blaudez and Chalot 2011; Bolchi et al. 2011; Colpaert et al. 2011).
Mechanisms involved in the intracellular detoxification or organellar compartmentation of heavy metals. A variety of mechanisms may be involved in transport phenomena contributing to decreased uptake and/or increased efflux. A variety of specific or non-specific mechanisms may also lead to intracellular chelation and intracellular precipitation, and to ROS detoxification
In ERM fungi, metal transporters involved in metal efflux were identified by Sharples et al. (2000b, 2001) in arsenate-resistant isolates of R. ericae. These isolates accumulated arsenate, which is a phosphate analogue and is transported by the phosphate uptake system. However, compared to isolates from unpolluted sites, R. ericae isolates from As/Cu mines had the capacity to reduce arsenate to arsenite, which was rapidly expelled from their mycelium via an increased efflux system (Sharples et al. 2000b, 2001).
Fungi may also decrease cytosolic free metal through the synthesis of a variety of metal-binding peptides and proteins. Among them, essential components of Cd detoxification pathways in various organisms are thiol compounds, including reduced glutathione, phytochelatins, and metallothioneins. Metallothioneins have been identified in most types of mycorrhizal fungi: they have been studied in both ECM fungi (Courbot et al. 2004; Bellion et al. 2006; Ramesh et al. 2009; Bolchi et al. 2011) and arbuscular mycorrhizal fungi (Lanfranco et al. 2002; González-Guerrero et al. 2007), where they were mostly found to be regulated. A cDNA coding for a Cu-metallothionein was found in the ERM fungus O. maius by Vallino et al. (2005), but gene expression was not affected by Zn treatment.
Thiol compounds such as glutathione and phytochelatins are major players in the anti-oxidative response in plants and in some fungi (Courbot et al. 2004; Yadav 2010). There is currently only very scanty information on the thiol-dependent antioxidant systems in ERM fungi, with the identification of a thioredoxin gene in the O. maius cDNA library (Vallino et al. 2005). By contrast, the complete thiol-dependent antioxidant systems of the ECM fungus Laccaria bicolor (Morel et al. 2008) and of Tuber melanosporum (Bolchi et al. 2011) have been recently characterised at the genome scale.
In addition to thiols compounds, fungi can protect themselves from the oxidative stress caused by heavy metals through the synthesis of enzymatic antioxidants such as catalase, peroxidase and superoxide dismutase (Jacob et al. 2001; Guelfi et al. 2003; Todorova et al. 2008). In O. maius Zn, two extracellular proteins showed high homology with superoxide dismutases (SOD), that play a protective role against free O2—radical toxicity, catalysing their conversion in hydrogen peroxide and oxygen (Fridovich 1995). Their induction by heavy metals has been described by several authors in plants, animals and micro-organisms (Chongpraditnun et al. 1992; Yoo et al. 1999; Vido et al. 2001). Treatment with high concentrations of zinc ions resulted in an increased amount and activity of both intracellular and extracellular SOD enzymes in an O. maius isolate from a polluted site (Martino et al. 2002; Vallino et al. 2009). The Cu/Zn SOD seems to be induced in O. maius specifically by zinc, as suggested by a comparative high-throughput proteomic investigation to elucidate common and specific responses of O. maius Zn to zinc and cadmium (Chiapello et al., unpublished data). The increased production of an extracellular Cu/Zn SOD, by binding metals and by performing its specific enzyme activity, could help both the fungus and the host plant to cope with the ROS formed in the extracellular medium, caused by metal pollution. Further demonstration of the important role of the Cu/Zn SOD enzyme in O. maius metal tolerance derived from heterologous expression of the corresponding gene in yeast (Vallino et al. 2009) and from the gene knock-out via homologous recombination and gene disruption (Abbà et al. 2009). Compared with the wild-type strain, the O. maius SOD1-null mutants showed a significant increase in zinc and cadmium sensitivity.
C. Omics Approaches to the Study of Heavy Metal Tolerance
Investigations on specific components (e.g. transporters, SODs, chelating metabolites) described in the previous paragraphs demonstrate that they may play significant roles in heavy metal tolerance in ERM fungi. However, this targeted approach often relies on existing knowledge acquired in other biological systems, and may miss specific mechanisms operating in the organisms under investigation, or general mechanisms so far unidentified. Untargeted approaches are therefore an important source of novel information, especially if they are supported by functional assays (Ruytinx et al. 2011).
The first attempt to investigate ERM fungal genes involved in zinc metal tolerance was through the sequencing of a small EST collection (Vallino et al. 2005). Variation in gene expression after treatment with high concentrations of Zn was monitored on 130 unigenes by reverse Northern blot hybridisation (Fig. 14.6): 16 unigenes were shown to be either up or down regulated. Among the differentially regulated genes, Vallino et al. (2005) could not find any previously reported heavy metal responsive or stress-related genes.
Reverse Northern blot hybridisation of part of the O. maius Zn EST unigenes identified by Vallino et al. (2005), with two probes obtained from mycelia grown for 20 days in the absence of Zn (C) or with addition of 10 mM Zn (T). EF Elongation factor 1α (housekeeping gene used for data normalisation), A absent signal (ratio signal/background <3), ⇑ up-regulated genes (ratio treated/control >3.0), ⇓ down-regulated genes (ratio treated/control <0.3), = not regulated genes (ratio treated/control between 0.3 and 3.0) (Modified from Vallino et al. (2005))
Most of the genes identified in the EST library and included in the cell defence category (e.g. Cu-metallothionein, Cu/Zn SOD, ascorbate peroxidase, thioredoxin, heat shock proteins) were not affected by the Zn treatment. This may be because the tolerant strain O. maius Zn (whose mRNA was used to construct the library and to hybridise the reverse Northern blots) does not perceive the metal concentration in the growth medium (10 mM Zn) as a serious stress condition, or because these genes may be involved at an earlier stage.
An untargeted approach using genomic microarray has been recently applied to Cadophora finlandica (Gorfer et al. 2009). C. finlandica, as described in Sect. II.C, can potentially associate with both ERM and ECM hosts (Vrålstad et al. 2002b). It is frequently found in heavy metal polluted habitats, and a possible functional role in heavy metal resistance by the host plants has been suggested (Vrålstad et al. 2002a). The genomic microarray analysis was conducted on a metal tolerant isolate of C. finlandica derived from an ECM Salix caprea root tip growing on a heavily contaminated soil (Dos Santos Utmazian et al. 2007) and compared the expression profiles on media supplemented with different metals (Gorfer et al. 2009). Many of the newly identified heavy metal regulated genes in C. finlandica encoded proteins of unknown functions or proteins with no established roles in heavy metal detoxification, but a large set of regulated genes were predicted to encode extracellular or plasma membrane proteins (e.g. transporters or secreted proteins). The majority of heavy metal defence activities in C. finlandica is therefore thought to take place outside the cell (Gorfer et al. 2009). It remains to be established if the C. finlandica isolate investigated in this study is truly mycorrhizal on ericaceous hosts.
Construction of cDNA libraries from ERM fungi growing on metal-containing medium, coupled with a functional assay in metal sensitive yeast mutants has allowed the identification of novel genes involved in metal tolerance in O. maius. In particular, a cDNA library obtained from O. maius growing on Cd was transformed in the S. cerevisiae cadmium-sensitive mutant yap1. Most yeast transformants able to grow on cadmium were found to harbour an insert coding for the same PLAC8 domain-containing protein, named OmFCR (O. maius fungal cadmium resistance) because of the sequence similarity with a plant protein involved in cadmium resistance (Abbà et al. 2011). The PLAC8 domain is widespread and evolutionary conserved in several proteins found in all eukaryotic kingdoms, but has no assigned biological role.
When tested on a number of different metals and stressful conditions, the OmFCR protein was found to confer specific resistance to cadmium. Direct measurement of cadmium in exposed yeast cells indicate no changes in this metal content caused by the presence of OmFCR (Abbà et al. 2011). This result would exclude that OmFCR is either a membrane efflux pump or a heavy metal chelator, as the expression of an efflux pump would decrease the cellular Cd content in the cell, while an intracellular chelator would increase it. The use of the yeast two-hybrid assay, followed by a number of experiments with specific yeast mutants illustrated by Abbà et al. (2011), indicates that OmFCR confers cadmium resistance to yeast cells through its interaction with Mlh3p, a subunit of the mismatch repair (MMR) system. The MMR pathway repairs base-base mismatches and insertion/deletion loops that arise from DNA duplication, as well as mismatches in heteroduplexes that are formed during recombination (Fishel and Kolodner 1995).
Cadmium is known to bind to the MMR system and to reduce its capacity to recognise small misalignments and base—base mismatches by disrupting its structure and function (Jin et al. 2003). Unlike many other genotoxic metal ions, cadmium, in fact, does not inflict direct damage on DNA, proteins and lipids through the generation of reactive oxygen species (ROS), but it targets proteins that are directly or indirectly involved in DNA repair and in antioxidant defence, altering their functions and ultimately causing toxic, mutagenic and carcinogenic effects. Although a first hypothesis could be a direct role of OmFCR in the repair of DNA damage caused by cadmium, the experiments described by Abbà et al. (2011) suggest that OmFCR may rather take part to the fairly unexplored role of the MMR system in connecting the recognition of DNA lesions with downstream signalling cascades that ultimately lead to cell cycle checkpoints (Fig. 14.7).
Working model for OmFCR. The genotoxic stress caused by cadmium might recruit the MMR system, which, in its turn, might promote the firing of OmFCR through protein—protein interactions with Mlh3p. The signalling pathway promoted by OmFCR appears to merge with the final part of the Mec1p-dependent phosphorylation cascade, at the Rad53p/Dun1p level. In pFL61-transformed cells (left) Dun1p is likely to recruit effector proteins that cause cell cycle arrest, while the presence of OmFCR (right) might enlist alternative effector proteins that ultimately allow the progression of cell division (Modified from Abbà et al. (2011))
V. Ericoid Mycorrhizal Fungi as Model Systems
Although mycorrhizae are critical elements of terrestrial ecosystems, we have just begun to understand the molecular interactions between mycorrhizal fungi and their host plants. The release of the complete Laccaria bicolor and Tuber melanosporum genomes (Martin et al. 2008, 2010) and the on-going genome sequencing of additional mycorrhizal fungi by the DOE Joint Genome Institute (http://www.jgi.doe.gov/) are going to provide unprecedented opportunities for studying the biology and the evolution underlying the symbiotic lifestyle. These new fungal genome sequencing programs proposed by Francis Martin within the international project “Exploring the Genome Diversity of Mycorrhizal Fungi to Understand the Evolution and Functioning of Symbiosis in Woody Shrubs and Trees” and the following comparison with various pathogenic and saprobic fungal genomes are likely to shed light on the complexity of plant—fungus associations and lead to the identification of the key gene set associated with mycorrhizal symbiosis.
Investigations on specific genes mediating symbiotic events in mycorrhiza formation have hitherto been based exclusively on the study of non-mycorrhizal (Myc-) plant mutants interacting with AM fungi (Parniske 2004). An equivalent development of molecular and genetic approaches is now required at the fungal side to obtain a complete picture of the symbiosis (Helber and Requena 2008). Gene function assignment will represent a critical step in the identification and the study of genes essential for symbiosis, but this procedure depends on the development of high-throughput methodologies to test gene function in vivo or in vitro. One of such approaches is the genetic transformation, i.e. the incorporation of exogenous DNA that causes the change or disruption of a gene followed by phenotype alterations.
The first evidence for genetic transformation of a mycorrhizal fungus was the successful transformation of the basidiomycete L. laccata using a protoplast-PEG-based transformation (Barrett et al. 1990).
Since then, other mycorrhizal species were stably transformed: several ectomycorrhizal (ECM) fungi, such as Hebeloma cylindrosporum (Marmeisse et al. 1992; Pardo et al. 2002; Combier et al. 2003), Paxillus involutus (Bills et al. 1995; Pardo et al. 2002), Laccaria bicolor (Bills et al. 1999; Kemppainen et al. 2005), Suillus bovinus (Hanif et al. 2002; Pardo et al. 2002) and Pisolithus tinctorius (Rodriguez-Tovar et al. 2005), and one ERM fungus, the ascomycete Oidiodendron maius strain Zn (Martino et al. 2007). However, arbuscular mycorrhizal fungi and other ECM fungi, such as Tuber borchii, seem to be recalcitrant to stable transformation (Forbes et al. 1998; Grimaldi et al. 2005; Helber and Requena 2008). All these genetic transformations led to the random integration of a selectable marker into the host genome. Random mutagenesis is a powerful approach to the creation of large collections of random mutants which can be then screened for a phenotype of interest. Combier and colleagues, for example, selected ten Myc-transformants blocked at the early stages of ectomycorrhiza formation from a H. cylindrosporum collection of random mutants, although the identification and the functional assignment of the mutated genes were not undertaken (Combier et al. 2004).
The most direct ways to explore gene function are generally the complete and stable inactivation of the target gene via homologous recombination at the wild-type locus (gene knock-out) or the down-regulation of its expression by the introduction of an antisense RNA molecule (gene knock-down). As demonstrated by Kemppainen et al. (2009), gene knock-down may represent a reliable alternative to disruption experiments for functional analysis of genes, especially in fungi with multinuclear hyphae, such as L. bicolor, or in fungi with a low frequency of homologous recombination. Yet, gene silencing does not ensure the complete suppression of the target gene expression as opposed to null mutants.
Although gene disruption in mycorrhizal fungi is limited by the high frequency of ectopic integrations of the transforming DNA molecule, the first targeted gene inactivation in a mycorrhizal fungus has proved to be a successful approach to study genes involved in the mycorrhization process (Abbà et al. 2009). The stable inactivation of the Cu/Zn superoxide dismutase (SOD1) gene by Agrobacterium-mediated transformation (AMT) has demonstrated, in fact, that in addition to a general role as an anti-oxidant enzyme, SOD1 is involved in the O. maius morphogenetic responses to the symbiotic partner.
SODs play a role in both pathogenic and symbiotic interactions between fungi and other organisms (Scott and Eaton 2008). An up-regulation of fungal ROS scavenger machinery during the early stages of the interaction has been demonstrated in pathogenic fungi (Hwang et al. 2002; Cox et al. 2003; Brown et al. 2008), as well as in arbuscular (Lanfranco et al. 2005) and ectomycorrhizal (Baptista et al. 2007) fungi. Cu/Zn SOD is the first fungal protein with a demonstrated role in the establishment of the ERM symbiosis, and the disruption of the corresponding gene is proposed to cause an imbalance in the redox homeostasis during host colonisation and an alteration in the delicate dialogue between O. maius and its host plant (Abbà et al. 2009).
Besides the possibility of gene deletion by homologous recombination, O. maius can be easily transformed by random insertional mutagenesis. A collection of more than 2000 random mutants is already available to be screened for the phenotype of interest and transformation with plasmid vectors for GFP tagging has been successfully used for protein localisation (Abbà, personal communication).
O. maius can be easily grown in vitro, where it reproduces asexually by forming conidia with just a single haploid nucleus. Moreover, the haploid monokaryotic status implies that the modifications of the wild-type phenotype, if detectable, can be observed even when mutations are recessive. This characteristic makes this fungus a good candidate to study mutants, because uninucleated spores can germinate and produce a homokaryotic mycelium with all the nuclei carrying the mutation.
Fungal genes potentially implicated in the mycorrhization process have been so far studied in the non-mycorrhizal filamentous fungus Magnaporthe oryzae (Heupel et al. 2010; Kloppholz et al. 2011). Although there are several types of mycorrhiza in terms of ecological significance and host—fungus association, a mycorrhizal fungus like O. maius might represent a more suitable heterologous system for studying fungal genes involved in the symbiosis. The feasibility of stable transformations and targeted gene disruptions in O. maius might open new possibilities to study the biological role of symbiosis-regulated genes, especially of those encoding effector-like secreted proteins, which have been hypothesised to interact with host plant proteins or alter their expression during the symbiosis interaction (Martin et al. 2008).
VI. Conclusions
A classical view is that distinct mycorrhizal types exist in nature, each with a well described morphology of the plant—fungus association (Smith and Read 1997) and a distinct range of symbiotic fungi. Although the classification into mycorrhizal types has been important to describe the diversity of symbiotic plant—fungus associations, it has lead to the view of a sharp separation among endo- and ectomycorrhizae. However, starting from the observation of common fungal taxa in ECM and ERM plants in nature (Bergero et al. 2000; Vrålstad et al. 2000, 2002a; Grelet et al. 2010), it was shown that the same fungus isolated from ECM roots could form ERM on ericaceous hosts and improve plant growth and/or nutrition (Villarreal-Ruiz et al. 2004; Grelet et al. 2009a). Although the range of potential interactions of ERM fungi is a delicate issue still open to debate, the observations on M. bicolor (Villarreal-Ruiz et al. 2004) indicate, at least for some ERM fungi, a phenotypic plasticity in their interactions with plants of different mycorrhizal status and contribute to weaken the boundaries between the classical mycorrhizal types. Similar conclusions also derive from studies on the taxonomic diversity of symbiotic fungi in other mycorrhizal symbioses. For example, well known ECM fungi have been found to associate with mycoheterotrophic and mixotrophic forest orchids, where they form typical endomycorrhizal coils. This phenotypic plasticity of ECM fungi in orchids has been demonstrated for both ascomycetes (e.g. Selosse et al. 2004) and basidiomycetes (e.g. Selosse et al. 2002; Girlanda et al. 2006).
There is increasing evidence that fungi taxonomically related to ERM fungi are common associates of plants with different mycorrhizal status (e.g. Chambers et al. 2008; Ohtaka and Narisawa 2008; Tedersoo et al. 2009), with a continuum from loose non-mycorrhizal to mycorrhizal associations between ericoid fungi and host plants, possibly related to environmental conditions and to the host plant species. It becomes now important to investigate whether these potential hyphal connections, irrespective of the morphology of fungus—plant interactions, do contribute to a functional mycelial network that may allow nutrient transfer in a mixed plant community, similar (or perhaps even connected) to the wood-wide web first demonstrated for ECM plants by Simard et al. (1997).
All ERM fungi appear to be well equipped with an arsenal of hydrolytic enzymes that are instrumental in the litter decomposition in heathlands as well as in woodlands.
Given the ECM potential of M. bicolor isolates, it is quite interesting that the genomic sequencing of the ECM fungus Laccaria bicolor indicates a strong reduction in the number of genes involved in the production of hydrolytic enzymes, as compared to saprotrophic fungi (Martin and Selosse 2008). However, it should be noted that some ECM forming ascomycetes, such as Sphaerosporella brunnea and Geopyxis carbonaria, that colonise seedlings particularly after fire, effectively degrade cellulose, produce phenol oxidase and degrade lignin (see Egger 2006). A genomic investigation of wider range of ERM and ECM fungi will be important to provide a more comprehensive understanding of the saprotrophic potential of these ecto- and endomycorrhizal fungi.
Biochemical, physiological and molecular studies have allowed us to gain better insights on the mechanisms of stress tolerance of ERM fungi, especially towards heavy metals (see Sect. IV). The identification of these mechanisms provides us with some clues to better understand the success observed for members of the “R. ericae aggregate” and O. maius in colonising heavy metal polluted soils. More mechanisms likely enable ERM fungi to protect themselves and their host plant from toxic compounds, and will hopefully emerge from genome wide approaches as well as from comparisons between metal tolerant and metal sensitive ERM strains (Murat et al. 2011).
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Perotto, S., Martino, E., Abbà, S., Vallino, M. (2012). 14 Genetic Diversity and Functional Aspects of Ericoid Mycorrhizal Fungi. In: Hock, B. (eds) Fungal Associations. The Mycota, vol 9. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-30826-0_14
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