Review
Ectomycorrhizas Ð extending the capabilities of rhizosphere
remediation?
Andrew A. Meharg
a, John W.G. Cairney
b,*
a
Department of Plant and Soil Science, University of Aberdeen, Cruickshank Building, St. Machar Drive, Aberdeen AB24 3UU, UK b
Mycorrhiza Research Group, School of Science, University of Western Sydney, P.O. Box 10, Kingswood NSW 2747, Australia
Accepted 20 March 2000
Abstract
The potential of ectomycorrhizal (ECM) associations to facilitate clean-up of soil contaminated with persistent organic pollutants (POPs) is considered. Most ECM fungi screened for degradation of POPs (e.g. polyhalogenated biphenyls, polyaromatic hydrocarbons, chlorinated phenols, and pesticides) are able to transform these compounds. Mineralization of toluene, tetrachloroethylene and 2,4-dichlorophenol in intact ECM-association rhizospheres has also been demonstrated. We review and consider the likely mechanisms by which ECM fungi can transform pollutants, the extent to which these capabilities may be utilized practically in bioremediation, along with the potential advantages and disadvantages of using ECM associations in bioremediation.72000 Elsevier Science Ltd. All rights reserved.
Keywords:Mycorrhizas; Persistent organic pollutants; Bioremediation; Phytoremediation
1. Introduction
Considerable attention has been focused on the po-tential use of plants to remediate soils contaminated with metal and persistent organic pollutants (POPs) (Anderson et al., 1993; Salt et al., 1998). Rhizosphere remediation technologies oer potentially cheap, low disturbance approaches to decontaminating polluted land. There is, however, concern regarding the time-scale required for successful plant-mediated remedia-tion (Anderson et al., 1993). Furthermore, although generally considered to be a clean technology, plant-based remediation is not without environmental impli-cations, particularly movement of contaminants into plants and so, potentially into wild-life food chains (Anderson et al., 1993).
Phytoremediation is widely applied as a catch-all term for the use of plants to remediate both
metal-and POP-contaminated soils. The term is certainly sui-ted to hyperaccumulation of metals by plants, since the plant tissues are the repository of the pollutants (Salt et al., 1998). Where plants are used to remediate POPs, however, we prefer the term `rhizosphere reme-diation', because POP degrading activity will, in most scenarios, occur in the rhizosphere, rather than in the plant per se.
Enhanced degradation or mineralization in the rhi-zosphere has been demonstrated for a range of pesti-cides, polyaromatic hydrocarbons (PAHs), oil, surfactants and chlorinated alkanes (Anderson et al., 1993). While it is thought that enhanced rhizosphere degradation is due to plant-stimulated microbial ac-tivity in the rhizosphere, other biological (e.g. bacterial plasmid transfer) and physical (e.g. pollutants drawn into the rhizosphere by the transpiration stream, alteration of soil structure) factors may also play a role. Rhizosphere microorganisms may not degrade POPs to yield energy, rather they may co-metabolize them as a consequence of utilizing plant-derived cyclic compounds. For example, plant phenolics, such as
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* Corresponding author. Tel.: 9903; fax: +61-2-9685-9915.
Table 1
Persistent organic pollutant degrading capabilities (+ orÿ)
afor a range of ectomycorrhizal fungi
Compoundsb Phe Ant Flu Pyr Per PCBa PCBb PCBc PCBd PCBe PCBf PCBg PCBh PCBi PCBj PFB TNT DCP Chl
Referencec 1 1 1 1 1 2d 2 2 2 2 2 2 2 2 2 3 4 5 6
Species
Amanita muscaria + + + + ÿ ÿ
A. rubescens ÿ + + + ÿ +
A. spissa + + + + +
Boletus grevellei ÿ
Bysporia terrestris + ÿ + ÿ ÿ ÿ ÿ ÿ + ±
Cenococcum geophilum ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ
Gautieria caudata ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ
G. crispa + + + + ÿ + ÿ ÿ ÿ ÿ
G. othii + + + ÿ ÿ + ÿ ÿ ÿ ÿ
Genabea cerebriformis
Hebeloma crustuliniforme + + + + + + ÿ ÿ ÿ + +
H. cylindrosporum +
H. hiemale + + + + + +
H. sarcophyllum +
H. sinapizans + + + + + ÿ
Hysterangium gardneri + + ÿ ÿ ÿ + + ÿ ÿ ÿ
Laccaria amethystina ÿ + + + +
Lactarius deliciosus + + + + +
L. deterrimus ÿ + + + ÿ
L. rufus ÿ + + + +
L. torminosus ÿ + + + +
Morchella conica + + + + +
M. elata + + + + +
M. esculenta + + + + +
Paxillus involutus + + + + + ÿ + +
Piloderma croceum + ÿ ÿ ÿ + + ÿ ÿ
Pisolithus tinctorius ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ + ÿ
Radiigera atrogleba + + + ÿ ÿ + + + +
Rhizopogon luteolus ÿ
R. roseolus +
R. vinicolor ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ
R. vulgaris ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ
Russula aeruginea ÿ ÿ ÿ ÿ ÿ
R. foetens ÿ + ÿ ÿ ÿ
Suillus bellini +
S. bovinus ÿ
S. granulatus + + + + + + + ÿ ÿ + + ÿ ÿ ÿ ÿ
S. luteus ÿ ÿ
S. variegatus + + + + + + + + +
Thelophora terrestris +
A.A.
Meharg,
J.W.G.
Cairney
/
Soil
Biology
&
Biochemis
try
32
(2000)
1475±14
catechin and coumarin serve as co-metabolites for degradation of polychlorinated biphenyls (PCBs) by bacteria (Salt et al., 1998). Enzymes used to degrade plant-derived compounds by free-living rhizosphere mi-crobial biomass may also degrade POPs. This is suggested, for example, by the results of Sanderman and Loos (1984), who isolated 2,4-Ddegrading bacteria
from the rhizospheres of sugarcane growing in soil that had not previously been exposed to the chemical. The full suite of enzymatic processes required to degrade a POP may not be possessed by a single organism. The rhizosphere comprises a complex con-sortium of microorganisms and POP degradation by rhizosphere consortia has been demonstrated by Lap-pin et al. (1995) for the pesticide mecoprop.
There are a number of important challenges to be considered in using plants to remediate POP-contami-nated sites. Such sites are rarely contamiPOP-contami-nated with only a single pollutant and the plant species used must be resistant to all contaminants present. Often indus-trial sites oer very poor habitats for plant growth due to poor nutritional status and soil structure. For rhizo-sphere remediation to be optimized, the surface area of root±soil contact must be considered, as this is a cru-cial factor in the eectiveness and speed of remedia-tion. For sites contaminated with multiple pollutants, the enzymatic activities of the organisms deployed to facilitate remediation need to be capable of degrading a wide range of POPs. These criteria, along with further potential remediation bene®ts, are met by
ecto-mycorrhizal (ECM) associations (Donnelly and
Fletcher, 1994), and it is the rhizosphere remediation potential of ECM associations that is considered in this review.
2. Why consider ECM associations for use in remediation?
A range of ECM fungi have been shown to degrade ®ve major classes of environmentally important POPs. Out of the 42 species of ECM fungi screened so far, 33 have been shown to degrade one or more classes of the chemicals (Table 1). Lower (2±3) chlorinated PCBs were readily degraded by eight out of the 13 species screened, while a limited number of the 4±5 chlori-nated biphenyls were degraded by only two species (Donnelly and Fletcher, 1995). Only one out of 21 species tested in a further study could not degrade at least one PAH, with over half degrading all ®ve PAHs to which they were exposed (Gramss et al., 1999). Interestingly, where a species could not degrade the full suite of PAHs with which it was challenged, there was a preference to degrade 4±5 ring PAHs, rather than the simpler three ring PAH structure. Some ECM fungi have also been shown to degrade chlorpropham
Tricho
(Rouillon et al., 1989), dichlorophenol (Meharg et al., 1997a), trinitrotoluene (Meharg et al., 1997b) and mono¯uorobiphenyl (Green et al., 1999). Although rates of degradation of some POPs by certain ECM fungi appear somewhat lower than those reported for some wood-rotting fungi (e.g. Gramms et al., 1999), we are mindful that the eciency of degradation will depend on a number of factors including growth rates of fungi, culture conditions, incubation time and nutri-ent. Some ECM fungi have, for example, been shown to remove up to 90% of trinitrotoluene (Meharg et al., 1997) and 95% of mono¯uorobiphenyl (Green et al., 1999) from solution culture. Similarly, up to 50% removal of PAHs, including for very recalcitrant com-pounds, such as benzo-(a)-pyrene from solution culture has been observed for some ECM taxa (Braun-Lulle-mann et al., 1999; Gramms et al., 1999). Importantly, all ECM fungal isolates tested to date have been obtained from unpolluted soils, suggesting that ECM fungi may express POP-degrading activities in their natural habitats. It is therefore, likely that the enzymes mediating POP degradation have a fundamental role in the normal ecology of ECM.
The ability to degrade POPs, while growing in sym-biosis with a host plant is clearly a central
consider-ation in using ECM fungi in remediation of
contaminated soils. To date, there have been only two investigations of degradation under aseptic conditions with an intact fungus±plant system. Sarand et al. (1999) found no evidence form-toluene degradation by
Suillus bovinus either in axenic culture or while grow-ing with a plant partner. Meharg et al. (1997a), how-ever, demonstrated that isolates of Suillus variegatus
and Paxillus involutus could mineralize 2,4-dichloro-phenol both in axenic culture and in symbiosis with
Pinus sylvestris. During growth in nutrient replete axe-nic culture P. involutus mineralized up to 17% of the substrate over 17 days. Mineralization rates were lower (<3% over 13 days) when the fungi were grown in symbiosis with P. sylvestris under nutrient-limiting conditions, but this was greater than when the fungi were grown in the absence of a host under the same conditions (<1% mineralized) (Meharg et al., 1997a). It is of interest also, that, while P. involutus minera-lized more 2,4-dichlorophenol than S. variegatus in nutrient replete culture, the reverse was true during symbiosis with the host, emphasizing the need to work with symbiotic systems. Furthermore, although amounts mineralized by the plant±fungus systems were relatively low, it must be noted that they were small closed systems that had been grown for ca. 6 months prior to addition of 2,4-dichlorophenol. Fungal myce-lia may have been largely inactive at this point, and higher rates of mineralization may occur with younger, more active mycelia.
In addition to direct interactions with pollutants,
ECM fungi may indirectly in¯uence their degradation in the rhizosphere via the mycorrhizosphere eect, whereby their mycelial systems in soil may in¯uence the structure and activities of soil microbial assem-blages (see Smith and Read, 1997). Although inter-actions between ECM fungi and other soil microbes are complex and are currently poorly-understood, it is known that bacterial communities can be markedly altered in the mycorrhizosphere compared to the rhizo-sphere of non-mycorrhizal roots (Rambelli, 1973) and that some mycorrhizosphere bacteria (mycorrhization helper bacteria) play a role in ECM fungal infection of roots (Garbaye, 1994). It is thus dicult to separate the activities of ECM fungi per se from those of the mycorrhizosphere biota when non-axenic experimental systems are used. For example, enhanced mineraliz-ation of tetrachloroethylene (TCE) from soil excavated from a chemical waste dump was observed in the intact, non-sterile, rhizosphere of Pinus taeda, with mineralization doubled in comparison to non-vege-tated soil (Anderson and Walton, 1995). This study also showed that very little TCE was retained in plant tissue, allaying fears of toxicant transfer via plant ma-terial to other trophic levels on contaminated sites. Anderson and Walton (1995) point out that the mycorrhizosphere eect may have been important in this study, making it dicult to assess the direct invol-vement of ECM fungi in degradation.
Limited information is available on the mechanisms of degradation of POPs by ECM fungi. Donnelly and Fletcher (1995), Gramss et al. (1999), and Meharg et al. (1997b) measured only disappearance of PCBs, PAHs and TNT, respectively, providing no infor-mation on the extent or pathways of degradation. Rouillon et al. (1989) revealed that a range of ECM taxa could hydrolyze the carbamate pesticide chlor-propham to the intermediate 3-chloroanaline. In the most comprehensive pathway study conducted on ECM fungi to date, Green et al. (1999) determined that a range of taxa sequentially hydroxylated the non-substituted ring of 4-¯uorobiphenyl. The isolates did not, however, cleave the aromatic ring.
3. Similarities between ECM and white rot fungi
to their lignicolous habit, lack speci®city for particular substrates, meaning that they can degrade a range of complex natural aromatic polymers along with aro-matic pollutants (Field et al., 1993; Barr and Aust, 1994). The fact that WRF express ligninolytic activity extracellularly considerably enhances their potential for bioremediation of soil. This is principally because the enzymes and reactive intermediates can diuse to soil surfaces where POPs with high anity for soil or-ganic matter are bound (Barr and Aust, 1994). They can thus access pollutants that are not available to other organisms, and greatly increase the volume of soil exposed to the organism. The extracellular nature of degradation also means that hyphae of WRF do not necessarily come directly into contact with the pol-lutants, enabling them to avoid toxicity, while still facilitating degradation. Furthermore, extracellular transformation of chemicals can decrease soil toxicity, enabling these fungi to withstand very high concen-trations of certain pollutants. For example, some WRF can withstand cyanide concentrations 100-fold greater than other soil organisms, facilitating mineral-ization of the compound via ligninolytic enzyme activi-ties (Barr and Aust, 1994).
ECM and wood-rotting fungi occupy the same for-est ecosystems, but contrast in their ecological func-tion. ECM are symbionts of tree roots, while white and brown rots are saprotrophs, degrading dead plant-derived materials. Both, however, are faced with simi-lar phenolic substrates (including certain lignin struc-tures) in their environment. WRF and, to a lesser extent, brown rot fungi are known to degrade lignin to access cellulose which is their primary growth substrate (Barr and Aust, 1994), while it is thought that some ECM fungi may also degrade lignin (at least partially) principally to mobilize mineral nutrients sequestered in soil organic matter (Bending and Read, 1997). ECM fungi are also known to express extracellular activities of key POP-degrading enzymes (see later), and this extracellular activity may confer the same bene®ts to ECM fungi as to WRF with respect to POP degra-dation.
4. Evidence for the enzymatic basis of POP metabolism by ECM fungi
Although only a limited suite of ECM fungi has so far been investigated, it is clear that they produce (see later) a range of enzymatic activities similar to wood-rotting fungi, that will allow at least partial metab-olism of soil organic compounds, and are known to degrade many POPs (Barr and Aust, 1994). Durall et al. (1994) demonstrated that several ECM taxa could obtain reduced carbon via degradation of cellulose and hemicellulose substrates in symbiosis withPseudotsuga
menziesii (Douglas ®r). The cellulolytic and hemicellu-lolytic enzyme activities required for, at least, partial degradation of these substrates have also been ident-i®ed in other ECM fungi (see Cairney and Burke, 1994, 1996). Entry et al. (1991), Bending and Read (1995), and Colpaert and van Tichelen (1996) have reported some decomposition of litter associated with symbiotic ECM mycelia in non-sterile soil systems and in the ®eld, supporting a potential role for ECM fungi in decomposition processes. With respect to complex aromatic material, Durall et al. (1994) found no evi-dence of degradation of humic polymers by ECM fungi in symbiosis with P. menziesii under axenic con-ditions. In contrast, several investigators have provided evidence that, while the abilities of some ECM fungi to degrade some compounds may be limited (e.g. Bending and Read, 1996, 1997), others may partially degrade lignin and phenolic compounds in axenic cul-ture (Trojanowski et al., 1984; Haselwandter et al., 1990; Griths and Caldwell, 1992; Bending and Read, 1997). The enzymatic basis for degradation of these substrates by ECM fungi is, however, less clearly resolved.
A range of phenol oxidizing enzyme activities, including tyrosinase (EC 1.14.18.1), catechol oxidase (EC 1.10.3.1), ascorbate oxidase (EC 1.10.3.3) and lac-case (EC 1.10.3.2) have been detected associated with symbiotic ECM mycelia in unsterile soil (Bending and Read, 1995; Colpaert and van Laere, 1996; Gramss, 1997; Timonen and Sen, 1998). Elevated amounts of non-speci®c peroxidase (EC 1.11.1.7) and manganese peroxidase (EC 1.11.1.13) activities have also been recorded for soils colonized by ECM fungal mycelia compared to equivalent uncolonized soil (Griths and Caldwell, 1992; Gramss, 1997). While clearly indicating that enzyme activities are associated with ECM myce-lia, it is not possible from these data to separate enzyme activities produced by the ECM fungi from those resulting from changes to the rhizosphere micro-¯ora that may be facilitated by the activities of the ECM hyphae or those that utilize hyphal biomass or exudates (see Sun et al., 1999) as substrates.
Since several ECM fungal taxa have been shown to partially mineralize lignin and dehydrogenative poly-mers of lignin monopoly-mers in axenic culture (Troja-nowski et al., 1984; Haselwandter et al., 1990), it has been widely assumed that this re¯ects an ability of the
ECM fungi concerned to produce peroxidative
enzymes including lignin peroxidase and manganese peroxidase (e.g. Griths and Caldwell, 1992; Cairney and Burke, 1994). There is, however, little evidence that ECM fungi produce such activities. Indeed Cair-ney and Burke (1998) have argued that the partial lig-nin degradation observed in radiorespirometric mineralization studies, along with putative positive reactions for ligninolytic activity in agar cultures of
ECM fungi (e.g. Griths and Caldwell, 1992; Gramss et al., 1998), can readily be explained by an alternative mechanism. In each of these cases the experimental medium contained 60±70mM Fe, which is sucient to generate hydroxyl radicals from H2O2 via the Fenton
reaction in a manner similar to brown rot fungi (Backa et al., 1992). ECM fungi, includingSuillus var-iegatus and Pisolithus sp., release H2O2 via
carbo-hydrate oxidase activities, facilitating hydroxyl radical production (Burke and Cairney, 1998). The hydroxyl radical is unable to catalyze cleavage ofb±1 andb±O± 4 bonds (Kirk et al., 1985), thus degradation of lignin
by this mechanism appears to be con®ned to
demethoxylation, ring hydroxylation and side-chain oxidation (Gierer et al., 1992). This is consistent with the relatively low rates of lignin mineralization observed for ECM fungi (Trojanowski et al., 1984; Haselwandter et al., 1990). Oxalic acid secreted by ECM fungi (Paris et al., 1996) may, as is the case with brown rot fungi, act as electron donor for the re-duction of Fe3+ to Fe2+, facilitating the Fenton reac-tion (Dutton and Evans, 1996; Burke and Cairney, 1998). Where assays for lignin peroxidase have been conducted so as to avoid potential Fe-related artifacts, no activity has been found for ECM fungi (Bending and Read, 1996; Burke and Cairney, 1998; Chambers et al., 1999). Manganese peroxidase activity has, how-ever, been measured, and a gene equivalent to that for a known isozyme from Phanerochete chrysosporium
identi®ed in the ECM fungus Tylospora ®brillosa
(Chambers et al., 1999). This may confer, on T. ®bril-losaat least, the ability to oxidize phenols and pheno-lic lignin substructures via catalysis of alkyl±phenyl and Ca±Cb cleavage, yielding quinones and
hydroxy-quinones (Hatakka, 1994). Clearly, activity of peroxi-dative enzyme activities in the rhizosphere would require H2O2as part of the catalytic cycle of oxidative
activity. ECM fungi are known to produce H2O2
during carbohydrate oxidation (Burke and Cairney, 1998; and see earlier), and may also be produced, along with superoxide radicals, by ECM fungi and other fungi during degradation of organic acids in the rhizosphere (Hofrichter et al., 1998) or by the activity of peroxidases themselves (Kuhad et al., 1997).
A variety of ECM fungi has been reported to pro-duce several other non-speci®c phenol oxidase activi-ties during growth in axenic culture (Bending and Read, 1996; GuÈnther et al., 1998; Kanunfre and Zan-can, 1998; Gramss et al., 1999). While the caveat regarding the culture media used may apply in some instances, there is clear evidence of laccase production by Thelephora terrestris (Kanunfre and Zancan, 1998) and of catechol oxidase by Lactarius controversus
(Bending and Read, 1996). Catechol oxidase catalyzes production of phenoxy radicals from catechol, while laccase can also catalyze one-electron oxidation of
phe-nols to phenoxy radicals, along with catalyzing demethoxylation and cleavage of aromatic rings (Kuhad et al., 1997). ECM fungi thus produce a range of enzyme activities known to transform a wide variety of organic pollutants, making them potentially useful organisms to facilitate bioremediation. It is further possible that in common with many saprotrophic fungi, some ECM fungi may express intracellular mul-tifunctional oxidases that may allow intracellular transformation of PAHs, although this remains to be demonstrated.
5. Importance of the mycorrhizosphere community
Although not all ECM fungi can degrade all con-taminants against which they have been screened, the majority of fungi appear to be able to degrade a range of contaminants. It is evident from Table 1, that only a limited number of ECM fungal taxa (and only single isolates of each) have been investi-gated with respect to their abilities to degrade or-ganic pollutants. Given that well in excess of 6000 ECM fungal species are likely to exist worldwide (Molina et al., 1992; Bougher, 1994) and that con-siderable physiological variation exists even between individual isolates of a single species (Cairney, 1999), only a fraction of the potential of ECM fungi to degrade pollutants has so far been deter-mined. In particular, many basidiomycetes in the Corticiaceae and Thelephoraceae, hitherto regarded largely as wood decomposers, may be widespread ECM-formers (Erland and Taylor, 1999) and may be a useful group for future research studies. Despite the fact that below-ground communities of ECM fungi are characteristically dominated by a small number of common taxa (e.g. Gehring et al., 1998; Jonsson et al., 1999b), up to 100 ECM mor-photypes (representing either species or genera) may be present in a single hectare of pure stand forest (e.g. Goodman and Trofymow, 1998; Jonsson et al., 1999a). The diverse nature of ECM communities may enhance remediation by ensuring a suite of taxa capable of expressing a range of enzyme activi-ties.
The mycorrhizosphere microbial community
catabolic genes for hydrocarbon degradation) formed on the surface of external hyphae. Degra-dation of m-toluate has also been observed in P. sylvestris±S. bovinus rhizospheres inoculated with a strain of Pseudomonas ¯uorescens that bore a tolu-ene-degrading plasmid (Sarand et al., 1999). ECM fungi have, however, also been show to reduce bac-terial activity in the vicinity of their mycelia under some circumstances (Olsson et al., 1996), so that both positive and potentially negative interactions between ECM fungi and soil bacteria must be con-sidered in this context. Clearly, this is an area where more detailed future research is required.
Green et al. (1999) demonstrated that while ECM fungi could sequentially hydroxylate a halogenated biphenyl ring, they were unable to cleave the ring. This hydroxylation step is a thermodynamically limiting step in biphenyl ring degradation. The hydroxylated metabolites, being more polar than the parent com-pounds, are more bioavailable. It may be that the ECM fungi perform important initial metabolic steps and so facilitate degradation of substituted biphenyls by other rhizosphere organisms that have the enzy-matic capacity to degrade the compounds further. Indeed, bacteria generally require biphenyl as a co-sub-strate to degrade halogenated biphenyls (Donnelly et al., 1994; Gilbert and Crowley, 1997). The presence of ECM fungi may negate the need for the presence of co-substrates.
6. Resistance of trees and ECM fungi to pollutants
For rhizosphere remediation to be tractable, plant species must be chosen that can withstand the harsh environments found at contaminated sites. Industrial sites that are the focus of bioremediation activities tend to be polluted with complex mixtures of inorganic and organic toxicants. For example, coal gas sites are contaminated with arsenic, cyanide, phenols and PAHs amongst other compounds (Thomas and Lester, 1994). Other common multiple contamination scenarios include historically polluted ®ne and bulk chemical manufacturing and storage sites, tanneries, explosive manufacture and disposal sites and land-®ll sites. Such sites are also generally characterized by poor soil struc-ture and potentially nutrient de®ciency. Many of the sites which may be considered for in-situ remediation technologies oer considerable challenges to plant colonization. Tree-ECM associations are ideal target organisms because key ECM host species readily colo-nize polluted soils of interest. Birch (Betula) species, for example, are adept at colonizing highly polluted industrial soils (Hartley et al., 1997; Cairney and Meharg, 1999). Importantly, their mycorrhizas also proliferate under such conditions. In an unpublished
study involving one of the authors, ECM diversity and infection levels were found to be as high on birch that had self-seeded at a coal gas site as they were at uncontaminated sites (Ridgeway et al., unpublished data). Although birch species are clearly important, other tree species, such as Scots pine (Pinus sylvestris), loblolly pine (Pinus taeda) and willow (Salix) spp., should also be considered. These species all reportedly self-seed at contaminated sites and maintain functional ECM associations (Walton and Anderson, 1990; Anderson et al., 1993; Cairney and Meharg, 1999).
The resistance of ECM associations to metals has been an area of considerable research focus (see Hart-ley et al., 1997; Meharg and Cairney, 1999 for compre-hensive reviews). The general conclusion from these studies is that ECM fungi and their hosts can be extre-mely resistant to metal contamination. Indeed, tree species naturally recolonize sites at which graminac-eous species are known to have evolved adaptive metal resistance. Whether tree species and their ECM fungal partners are constitutively or adaptively resistant to metals is a subject of some speculation, with evidence that both strategies are employed to colonize contami-nated sites (Meharg and Cairney, 1999). Importantly, ECM diversity and infection levels are maintained in multiple contaminated sites (Cairney and Meharg, 1999).
POP resistance of ECM associations has received less attention (Cairney and Meharg, 1999). Nicolotti and Egli (1998) found that additions of crude oil could both stimulate and inhibit ECM infection on Picea abies and Populus nigra, depending on the concen-trations added, along with the time before addition or planting. They also showed that oil addition altered ECM fungal community structure, inhibiting some morphotypes, having neutral eect on others, and a stimulatory eect on the remainder. Some morpho-types were only observed at the highest concentrations; at which these became dominant, infecting over half the root tips on some plants. Sarand et al. (1999) showed that a Pinus sylvestris±Suillus bovinus associ-ation could withstand the presence of 2% w/v toluene with no ill eects when growing in an expanded clay medium. Similarly, when Pinus sylvestris±Suillus bovi-nus or Paxillus involutus associations where grown in the presence of petroleum hydrocarbons, no adverse eect was shown on hyphal or plant development (Sar-and et al., 1998).
7. Root structure of host-ECM fungus associations
Tree species have two major advantages over other plants when it comes to rhizosphere remediation: they produce considerable root biomass and many species are deep rooted. ECM fungi are known to produce
extensive extramatrical mycelial systems in forest soils. In some cases individual mycelia may exceed 300 m2 (e.g. Bonello et al., 1998; Sawyer et al., 1999). These mycelia are largely con®ned to the fermentation hor-izons, where they occupy a signi®cant soil volume (Leake and Read, 1997). The surface area of extrama-trical mycelium in mature forest soils is dicult to esti-mate, however, total mycelial lengths associated with pine and willow seedlings are up to 8000 times greater than the host root systems (Read and Boyd, 1986; Jones et al., 1990; Rousseau et al., 1994) and may increase the surface area up to 47-fold (Smith and Read, 1997). The volume of soil explored is thus con-siderable, and will be further enhanced by extracellular ECM fungus-derived enzyme activities. Trees display high rates of transpiration, a phenomenon that is exploited in the use of tree planting to `retain' con-taminated plumes around land-®ll sites (Cunningham et al., 1996). The transpiration stream may mobilize water soluble and semi-soluble pollutants into the rhi-zosphere, which is an ideal scenario with respect to bioremediation strategies. A further bene®t to using trees is that their growth will disturb the soil pro®le, generating cracks and root channels that both aerate the soil and act as conduits for venting of volatile and semi-volatile contaminants from deep within the soil pro®le (Cunningham et al., 1996).
8. Conclusions
In this review we have highlighted the potential for using ECM fungi and their host trees in remediation of soils contaminated by POPs and drawn compari-sons with the potential use of WRF. ECM fungi appear to have advantages and disadvantages over their white rot counterparts with respect to soil reme-diation. Inocula of WRF can be seeded onto sites using infected wood chips or vegetable materials (Barr and Aust, 1994), and inoculum density can be regu-lated. Although, depending on the site history, old roots, fallen branches etc. may already be present and aid the spread of WRF. Thus, if conditions are right, remediation may proceed relatively rapidly (Lamar et al., 1994). Remediation using ECM fungi will be slower, and will rely upon the development of tree root systems and associated fungal biomass. However, unlike white rot fungi, ECM fungi obtain a carbon supply from their tree hosts, meaning that once estab-lished, infection can be sustainable. The persistence of particular fungal taxa in root systems may depend on local soil conditions and activities of indigenous ECM fungi (Villeneuve et al., 1991), but there is good evi-dence that some introduced ECM fungi may persist for several years (Selosse et al., 1999). Furthermore, ECM fungal biomass in forest soils is considerable,
and roots with their associated mycelia will actively explore all available soil. In contrast, mycelia of WRF will remain largely where they are seeded. The speed at which a site needs to be remediated may be a deciding factor between the use of these two technologies. Indeed, where rapid remediation is required, direct ap-plication of xenobiotic-degrading bacteria (see Single-ton, 1994) may be a more useful alternative.
Clearly, only a limited number of ECM fungi have been screened against a limited number of POPs and wider screening is required. Few studies have demon-strated mineralization of POPs, thus the chemical fate of compounds requires further investigation. There is also an urgent need to scale up testing to large micro-cosm and ®eld trials, and detect and select appropriate host plant species. The criteria for such selection needs to be carefully considered in terms of speed at which bioremediation has to proceed, the suitability of the soil for tree growth and the contaminants present on a particular site. Management of trees (fertilization, cop-picing, irrigation) may also have to be considered to optimize remediation. Use of bioremediation technol-ogies is still in its infancy, with few examples of practi-cal application. Given pressures to use sustainable technologies, increasing restrictions on ex-situ disposal of contaminated soil, and the drive to clean-up poten-tially valuable agricultural or amenity sites, there is considerable impetus to develop technologies, such as rhizosphere remediation. Their importance to tree growth, abilities to produce extensive mycelia and key extracellular enzymes, along with potential interactions with other soil microorganisms, suggest ECM fungi as key components in the future of rhizosphere remedia-tion technology.
Acknowledgements
We thank the anonymous referees for their construc-tive comments on the original draft of this review.
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