Ectomycorrhizas — extending the capabilities of rhizosphere remediation?

Soil Biology & Biochemistry 32 (2000) 1475±1484

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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. 7 2000 Elsevier Science Ltd. All rights reserved. Keywords: Mycorrhizas; Persistent organic pollutants; Bioremediation; Phytoremediation

1. Introduction Considerable attention has been focused on the potential 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 o€er potentially cheap, low disturbance approaches to decontaminating polluted land. There is, however, concern regarding the timescale required for successful plant-mediated remediation (Anderson et al., 1993). Furthermore, although generally considered to be a clean technology, plantbased remediation is not without environmental implications, 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* Corresponding author. Tel.: +61-2-9685-9903; fax: +61-2-96859915. E-mail address: [email protected] (J.W.G. Cairney).

and POP-contaminated soils. The term is certainly suited 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 remediation', 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 rhizosphere has been demonstrated for a range of pesticides, 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 activity 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

0038-0717/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 8 - 0 7 1 7 ( 0 0 ) 0 0 0 7 6 - 6

+ + +

+

+

+

+ + + + + + + + +

ÿ +

+

+

+

+

+

ÿ + ÿ ÿ ÿ + + + +

ÿ ÿ

+

+

1

Ant

+ ÿ +

1

Referencec

Amanita muscaria A. rubescens A. spissa Boletus grevellei Bysporia terrestris Cenococcum geophilum Gautieria caudata G. crispa G. othii Genabea cerebriformis Hebeloma crustuliniforme H. cylindrosporum H. hiemale H. sarcophyllum H. sinapizans Hysterangium gardneri Laccaria amethystina Lactarius deliciosus L. deterrimus L. rufus L. torminosus Morchella conica M. elata M. esculenta Paxillus involutus Piloderma croceum Pisolithus tinctorius Radiigera atrogleba Rhizopogon luteolus R. roseolus R. vinicolor R. vulgaris Russula aeruginea R. foetens Suillus bellini S. bovinus S. granulatus S. luteus S. variegatus Thelophora terrestris

Species

Phe

Compoundsb

+

+

ÿ ÿ

+ + + + + + + + +

+

+

+

+ + +

1

Flu

+

+

ÿ ÿ

+ + + + + + + + +

+

+

+

+ + +

1

Pyr

+

+

ÿ ÿ

+ + ÿ + + + + + +

+

+

+

ÿ ÿ +

1

Per

+

+

+ ÿ ÿ + +

2d

PCBa

ÿ + ÿ ÿ

+ ÿ + ÿ ÿ

+

ÿ

ÿ

+ ÿ ÿ + +

2

PCBc

+

ÿ ÿ ÿ + +

2

PCBb

Table 1 Persistent organic pollutant degrading capabilities (+ or ÿ)a for a range of ectomycorrhizal fungi

+

ÿ ÿ

ÿ ÿ +

ÿ

ÿ

ÿ ÿ ÿ + ÿ

2

PCBd

ÿ

ÿ ÿ

ÿ ÿ ÿ

ÿ

ÿ

ÿ ÿ ÿ ÿ ÿ

2

PCBe

ÿ

ÿ ÿ

ÿ ÿ ÿ

+

+

ÿ ÿ ÿ + +

2

PCBf

+

ÿ ÿ

+ ÿ +

+

+

ÿ ÿ ÿ ÿ ÿ

2

PCBg

+

ÿ ÿ

+ ÿ +

ÿ

ÿ ÿ ÿ ÿ ÿ

2

PCBh

ÿ

ÿ ÿ

ÿ ÿ +

ÿ

+ ÿ ÿ ÿ ÿ

2

PCBi

ÿ

ÿ ÿ

ÿ ÿ +

ÿ

± ÿ ÿ ÿ ÿ

2

PCBj

ÿ ÿ + +

ÿ

3

PFB

+

+

+

4

TNT

+

+

5

DCP

+ ÿ ÿ ÿ +

ÿ +

ÿ

+ + + ÿ

ÿ

ÿ

ÿ +

6

Chl

1476 A.A. Meharg, J.W.G. Cairney / Soil Biology & Biochemistry 32 (2000) 1475±1484

The symbol `+' indicates that compounds were degraded to some extent. In some cases this was only a small % of the total available, but in others degradation was signi®cant. See text and original references for more details. b Compound codes: Phe, phenanthrene; Ant, anthracene; Flu, ¯uronthene; Pyr, pyrene; Per, perylene; PCBa, PCB-2,3; PCBb, PCB-2,2 '; PCBc, PCB-2,4 '; PCBd, PCB-4,4 '; PCBe, PCB-2,4,4 '; PCBf, PCB-2,5,2 '; PCBg, PCB-2,5,4 '; PCBh, PCB-2,4,2 ',4 '; PCBi, PCB-2,5,2 ',5 '; PCBj, PCB-2,4,6,2 ',4 '; PFB, 4-¯uorobiphenyl; TNT, trinitrotoluene; DCP, 2,4-dichlorophenol; Chl, chlorpropham. c References: 1 = Gramss et al. (1999); 2 = Donnelly and Fletcher (1995); 3 = Green et al. (1999); 4 = Meharg et al. (1997a); 5 = Meharg et al. (1997b); 6 = Rouillon et al. (1989). d Only PCBs that were degraded by at least one taxon are reported. A further 9 (4±6 chlorinated) PCBs were tested by Donnelly and Fletcher (1995), but were not degraded by any of the fungi tested.

a

Tricholoma lascivum T. terreum Tylospora ®brillosa

+ +

+ +

+ +

+ +

+ +

+

+

A.A. Meharg, J.W.G. Cairney / Soil Biology & Biochemistry 32 (2000) 1475±1484

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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 microbial biomass may also degrade POPs. This is suggested, for example, by the results of Sanderman and Loos (1984), who isolated 2,4-D degrading 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 consortium of microorganisms and POP degradation by rhizosphere consortia has been demonstrated by Lappin et al. (1995) for the pesticide mecoprop. There are a number of important challenges to be considered in using plants to remediate POP-contaminated sites. Such sites are rarely contaminated with only a single pollutant and the plant species used must be resistant to all contaminants present. Often industrial sites o€er very poor habitats for plant growth due to poor nutritional status and soil structure. For rhizosphere remediation to be optimized, the surface area of root±soil contact must be considered, as this is a crucial factor in the e€ectiveness and speed of remediation. 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 ectomycorrhizal (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 chlorinated 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

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(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 eciency of degradation will depend on a number of factors including growth rates of fungi, culture conditions, incubation time and nutrient. 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 compounds, such as benzo-(a)-pyrene from solution culture has been observed for some ECM taxa (Braun-Lullemann 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 symbiosis with a host plant is clearly a central consideration 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 for m-toluene degradation by Suillus bovinus either in axenic culture or while growing with a plant partner. Meharg et al. (1997a), however, demonstrated that isolates of Suillus variegatus and Paxillus involutus could mineralize 2,4-dichlorophenol both in axenic culture and in symbiosis with Pinus sylvestris. During growth in nutrient replete axenic 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 mineralized 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 mycelia 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 e€ect, whereby their mycelial systems in soil may in¯uence the structure and activities of soil microbial assemblages (see Smith and Read, 1997). Although interactions 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 rhizosphere 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 dicult 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 mineralization 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-vegetated 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 material to other trophic levels on contaminated sites. Anderson and Walton (1995) point out that the mycorrhizosphere e€ect may have been important in this study, making it dicult to assess the direct involvement 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 information on the extent or pathways of degradation. Rouillon et al. (1989) revealed that a range of ECM taxa could hydrolyze the carbamate pesticide chlorpropham 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 White rot fungi (WRF) are regarded as potentially excellent organisms for bioremediation since they produce a suite of extracellular oxidative lignin and humic acid degrading enzymes (Barr and Aust, 1994). The most studied of these are lignin peroxidase (EC 1.11.1.14), manganese peroxidase (EC 1.11.1.13) and laccase (EC 1.10.3.2), with di€erent taxa known to produce di€erent combinations of these (Hatakka, 1994). The oxidative activities and associated free radical intermediates produced by WRF, although central

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to their lignicolous habit, lack speci®city for particular substrates, meaning that they can degrade a range of complex natural aromatic polymers along with aromatic 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 di€use to soil surfaces where POPs with high anity for soil organic 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 pollutants, 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 concentrations of certain pollutants. For example, some WRF can withstand cyanide concentrations 100-fold greater than other soil organisms, facilitating mineralization of the compound via ligninolytic enzyme activities (Barr and Aust, 1994). ECM and wood-rotting fungi occupy the same forest ecosystems, but contrast in their ecological function. ECM are symbionts of tree roots, while white and brown rots are saprotrophs, degrading dead plantderived materials. Both, however, are faced with similar phenolic substrates (including certain lignin structures) 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 degradation. 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 woodrotting fungi, that will allow at least partial metabolism 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 with Pseudotsuga

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menziesii (Douglas ®r). The cellulolytic and hemicellulolytic enzyme activities required for, at least, partial degradation of these substrates have also been identi®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 evidence of degradation of humic polymers by ECM fungi in symbiosis with P. menziesii under axenic conditions. 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 culture (Trojanowski et al., 1984; Haselwandter et al., 1990; Griths 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 laccase (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 (Gri€ths and Caldwell, 1992; Gramss, 1997). While clearly indicating that enzyme activities are associated with ECM mycelia, 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 polymers of lignin monomers in axenic culture (Trojanowski 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. Griths and Caldwell, 1992; Cairney and Burke, 1994). There is, however, little evidence that ECM fungi produce such activities. Indeed Cairney and Burke (1998) have argued that the partial lignin degradation observed in radiorespirometric mineralization studies, along with putative positive reactions for ligninolytic activity in agar cultures of

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ECM fungi (e.g. Griths 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±70 mM Fe, which is sucient to generate hydroxyl radicals from H2O2 via the Fenton reaction in a manner similar to brown rot fungi (Backa et al., 1992). ECM fungi, including Suillus variegatus and Pisolithus sp., release H2O2 via carbohydrate oxidase activities, facilitating hydroxyl radical production (Burke and Cairney, 1998). The hydroxyl radical is unable to catalyze cleavage of b±1 and b±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 reduction of Fe3+ to Fe2+, facilitating the Fenton reaction (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, however, 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. ®brillosa at least, the ability to oxidize phenols and phenolic lignin substructures via catalysis of alkyl±phenyl and Ca ±Cb cleavage, yielding quinones and hydroxyquinones (Hatakka, 1994). Clearly, activity of peroxidative enzyme activities in the rhizosphere would require H2O2 as 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 produce several other non-speci®c phenol oxidase activities during growth in axenic culture (Bending and Read, 1996; GuÈnther et al., 1998; Kanunfre and Zancan, 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 multifunctional 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 contaminants 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 investigated with respect to their abilities to degrade organic pollutants. Given that well in excess of 6000 ECM fungal species are likely to exist worldwide (Molina et al., 1992; Bougher, 1994) and that considerable 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 determined. 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 morphotypes (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 activities. The mycorrhizosphere microbial community (including non-symbiotic fungi and bacteria) may act in concert with ECM fungi to degrade POPs (Sarand et al., 1999). Co-operative degradation of pollutants has certainly been demonstrated for rhizosphere bacterial communities in the absence of ECM fungi (Lappin et al., 1985). Sarand et al. (1998) showed that when Pinus sylvestris±Suillus bovinus or Paxillus involutus associations were grown in soil contaminated with petroleum hydrocarbons, bacterial bio-®lms (which harboured plasmid-borne

A.A. Meharg, J.W.G. Cairney / Soil Biology & Biochemistry 32 (2000) 1475±1484

catabolic genes for hydrocarbon degradation) formed on the surface of external hyphae. Degradation of m-toluate has also been observed in P. sylvestris±S. bovinus rhizospheres inoculated with a strain of Pseudomonas ¯uorescens that bore a toluene-degrading plasmid (Sarand et al., 1999). ECM fungi have, however, also been show to reduce bacterial 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 considered 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 compounds, 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 enzymatic capacity to degrade the compounds further. Indeed, bacteria generally require biphenyl as a co-substrate 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 structure and potentially nutrient de®ciency. Many of the sites which may be considered for in-situ remediation technologies o€er considerable challenges to plant colonization. Tree-ECM associations are ideal target organisms because key ECM host species readily colonize 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

1481

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 Hartley et al., 1997; Meharg and Cairney, 1999 for comprehensive reviews). The general conclusion from these studies is that ECM fungi and their hosts can be extremely resistant to metal contamination. Indeed, tree species naturally recolonize sites at which graminaceous 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 contaminated 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 concentrations 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 e€ect on others, and a stimulatory e€ect on the remainder. Some morphotypes 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 association could withstand the presence of 2% w/v toluene with no ill e€ects when growing in an expanded clay medium. Similarly, when Pinus sylvestris±Suillus bovinus or Paxillus involutus associations where grown in the presence of petroleum hydrocarbons, no adverse e€ect was shown on hyphal or plant development (Sarand 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

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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 horizons, where they occupy a signi®cant soil volume (Leake and Read, 1997). The surface area of extramatrical mycelium in mature forest soils is dicult to estimate, 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 considerable, 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' contaminated plumes around land-®ll sites (Cunningham et al., 1996). The transpiration stream may mobilize water soluble and semi-soluble pollutants into the rhizosphere, 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 comparisons with the potential use of WRF. ECM fungi appear to have advantages and disadvantages over their white rot counterparts with respect to soil remediation. Inocula of WRF can be seeded onto sites using infected wood chips or vegetable materials (Barr and Aust, 1994), and inoculum density can be regulated. 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 established, 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 evidence 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 application of xenobiotic-degrading bacteria (see Singleton, 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 demonstrated mineralization of POPs, thus the chemical fate of compounds requires further investigation. There is also an urgent need to scale up testing to large microcosm 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, coppicing, irrigation) may also have to be considered to optimize remediation. Use of bioremediation technologies is still in its infancy, with few examples of practical application. Given pressures to use sustainable technologies, increasing restrictions on ex-situ disposal of contaminated soil, and the drive to clean-up potentially 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 remediation technology.

Acknowledgements We thank the anonymous referees for their constructive comments on the original draft of this review.

References Anderson, T.A., Guthrie, E.A., Walton, B.T., 1993. Bioremediation in the rhizosphere. Environmental Science and Technology 27, 2630±2636. Anderson, T.A., Walton, B.T., 1995. Comparative fate of [14C]trichloroethylene in the root zone of plants from a former solvent disposal site. Environmental Toxicology and Chemistry 14, 2041±2047. Backa, S., Gierer, J., Reitberger, T., Nilsson, T., 1992. Hydroxyl radical activity in brown-rot fungi studied by a new chemiluminescence method. Holzforschung 46, 61±67. Barr, D.P., Aust, S.D., 1994. Mechanisms white rot fungi use to degrade pollutants. Environmental Science and Technology 28, 79±87. Bending, G.D., Read, D.J., 1995. The structure and function of the

A.A. Meharg, J.W.G. Cairney / Soil Biology & Biochemistry 32 (2000) 1475±1484 vegetative mycelium of ectomycorrhizal plants. VI. Activities of nutrient mobilizing enzymes in birch litter colonized by Paxillus involutus (Fr.) Fr. New Phytologist 130, 401±409. Bending, G.D., Read, D.J., 1996. E€ects of the soluble polyphenol tannic acid on the activities of ericoid and ectomycorrhizal fungi. Soil Biology & Biochemistry 28, 1595±1602. Bending, G.D., Read, D.J., 1997. Lignin and soluble phenolic degradation by ectomycorrhizal and ericoid mycorrhizal fungi. Mycological Research 101, 1348±1354. Bonello, P., Bruns, T.D., Gardes, M., 1998. Genetic structure of a natural population of the ectomycorrhizal fungus Suillus pungens. New Phytologist 138, 533±542. Bougher, N.L., 1994. Diversity of ectomycorrhizal fungi associated with eucalypts in Australia. In: Brundrett, M., Dell, B., Malajczuk, N., Mingqin, G. (Eds.), Mycorrhizas for Plantation Forestry in Asia. Australian Centre for International Agricultural Research, Canberra, pp. 8±15. Braun-Lullemann, A., Huttermann, A., Majcherczyk, A., 1999. Screening of ectomycorrhizal fungi for degradation of polycyclic aromatic hydrocarbons. Applied Microbiology and Biotechnology 53, 127±132. Burke, R.M., Cairney, J.W.G., 1998. Carbohydrate oxidases in ericoid and ectomycorrhizal fungi: a possible source of Fenton radicals during the degradation of lignocellulose. New Phytologist 139, 637±645. Cairney, J.W.G., 1999. Intraspeci®c physiological variation: implications for understanding functional diversity in ectomycorrhizal fungi. Mycorrhiza 9, 125±135. Cairney, J.W.G., Burke, R.M., 1994. Fungal enzymes degrading plant cell walls: their possible signi®cance in the ectomycorrhizal symbiosis. Mycological Research 98, 1345±1346. Cairney, J.W.G., Burke, R.M., 1996. Plant cell wall-degrading enzymes in ericoid and ectomycorrhizal fungi. In: Azcon-Aguilar, C., Barea, J.M. (Eds.), Mycorrhizas in Integrated Systems. European Commission, Brussels, pp. 218±221. Cairney, J.W.G., Burke, R.M., 1998. Do ecto- and ericoid mycorrhizal fungi produce peroxidase activities? Mycorrhiza 8, 61±65. Cairney, J.W.G., Meharg, A.A., 1999. In¯uences of anthropogenic pollution on mycorrhizal fungal communities. Environmental Pollution 106, 169±182. Chambers, S.M., Burke, R.M., Brooks, P.R., Cairney, J.W.G., 1999. Molecular and biochemical evidence for manganese-dependent peroxidase activity in Tylospora ®brillosa. Mycological Research 103, 1098±1102. Colpaert, J.V., Van Laere, A., 1996. A comparison of the extracellular enzyme activities of two ectomycorrhizal and leaf-saprotrophic basidiomycetes colonizing beech leaf litter. New Phytologist 134, 133±141. Colpaert, J.V., Van Tichelen, K.V., 1996. Decomposition, nitrogen and phosphorus mineralization from beech leaf litter colonized by ectomycorrhizal or litter-decomposing basidiomycetes. New Phytologist 134, 123±132. Cunningham, S.D., Anderson, T.A., Schwab, A.P., Hsu, F.C., 1996. Phytoremediation of soils contaminated with organic pollutants. Advances in Agronomy 56, 55±114. Donnelly, P.K., Fletcher, J.S., 1994. Potential use of mycorrhizal fungi as bioremediation agents. In: Anderson, T.A., Coats, J.R. (Eds.), Bioremediation Through Rhizosphere Technology. American Chemical Society, Washington DC, pp. 93±99. Donnelly, P.K., Fletcher, J.S., 1995. PCB metabolism by ectomycorrhizal fungi. Bulletin of Environmental Contamination and Toxicology 54, 507±513. Donnelly, P.K., Hedge, R.S., Fletcher, J.S., 1994. Growth of PCBdegrading bacteria on compounds from photosynthetic plants. Chemosphere 28, 981±988. Durall, D.M., Todd, A.W., Trappe, J.M., 1994. Decomposition of

14

1483

C-labelled substrates by ectomycorrhizal fungi in association with Douglas ®r. New Phytologist 127, 725±729. Dutton, M.V., Evans, C.S., 1996. Oxalate production by fungi: its role in pathogenicity and ecology in the soil environment. Canadian Journal of Microbiology 42, 881±895. Entry, J., Rose, C.L., Cromack, K., 1991. Litter decomposition and nutrient release in ectomycorrhizal mat soils of a Douglas-®r ecosystem. Soil Biology & Biochemistry 23, 285±290. Erland, S., Taylor, A.F.S., 1999. Resupinate ectomycorrhizal fungal genera. In: Cairney, J.W.G., Chambers, S.M. (Eds.), Ectomycorrhizal Fungi: Key Genera in Pro®le. Springer-Verlag, Berlin, pp. 347±363. Field, J.A., de Jong, E., Feijoo-Costa, G., de Bont, J.A.M., 1993. Screening for ligninolytic fungi applicable to the biodegradation of xenobiotics. Trends in Biotechnology 11, 44±49. Garbaye, J., 1994. Helper bacteria: a new dimension to the mycorrhizal symbiosis. New Phytologist 128, 197±210. Gehring, C.A., Theimer, T.C., Whitham, T.G., Keim, P., 1998. Ectomycorrhizal fungal community structure of pinyon pine communities growing in two environmental extremes. Ecology 79, 1562±1572. Gierer, J., Yang, E., Reitberger, T., 1992. The reactions of hydroxyl radicals with aromatic rings in lignins, studied with creosol and 4-methylveratrol. Holzforschung 46, 495±504. Gilbert, E.S., Crowley, D.E., 1997. Plant compounds that induce polychlorinated biphenyl biodegradation by Arthrobacter sp. strain B1B. Applied and Environmental Microbiology 63, 1933± 1938. Goodman, D.M., Trofymow, J.A., 1998. Comparison of communities of ectomycorrhizal fungi in old-growth and mature stands of Douglas-®r at two sites in southern Vancouver Island. Canadian Journal of Forest Research 28, 574±581. Gramss, G., 1997. Activity of oxidative enzymes in fungal mycelia from grassland and forest soils. Journal of Basic Microbiology 37, 407±423. Gramss, G., GuÈnther, Th., Fritsche, W., 1998. Spot tests for oxidative enzymes in ectomycorrhizal, wood- and litter decaying fungi. Mycological Research 102, 67±72. Gramss, G., Kirsche, B., Voigt, K.-D., GuÈnther, Th., Fritsche, W., 1999. Conversion rates of ®ve polycyclic aromatic hydrocarbons in liquid cultures in ®fty-eight fungi and the concomitant production of oxidative enzymes. Mycological Research 103, 1009± 1018. Green, N.A., Meharg, A.A., Till, C., Troke, J., Nicholson, J.K., 1999. Degradation of 4-¯uorobiphenyl by mycorrhizal fungi as determined by 19F nuclear magnetic resonance spectroscopy and 14 C radiolabelling analysis. Applied and Environmental Microbiology 65, 4021±4027. Griths, R.P., Caldwell, B.A., 1992. Mycorrhizal mat communities in forest soil. In: Read, D.J., Lewis, D.H., Fitter, A.H., Alexander, I.J. (Eds.), Mycorrhizas in Ecosystems. CAB International, Wallingford, pp. 98±105. GuÈnther, Th., Perner, B., Gramss, G., 1998. Activities of phenol oxidising enzymes of ectomycorrhizal fungi in axenic culture and in symbiosis with Scots pine (Pinus sylvestris L). Journal of Basic Microbiology 38, 197±206. Hartley, J., Cairney, J.W.G., Meharg, A.A., 1997. Do ectomycorrhizal fungi exhibit adaptive tolerance to potentially toxic metals in the rhizosphere? Plant and Soil 189, 303±319. Haselwandter, K., Bobleter, O., Read, D.J., 1990. Degradation of 14 C-labelled lignin and dehydropolymer of coniferyl alcohol by ericoid and ectomycorrhizal fungi. Archives of Microbiology 153, 352±354. Hatakka, A., 1994. Lignin-modifying enzymes from selected whiterot fungi: production and role in lignin degradation. FEMS Microbiology Reviews 13, 125±135. Hofrichter, M., Ziegenhagen, D., Vares, T., Friedrich, M., Jager,

1484

A.A. Meharg, J.W.G. Cairney / Soil Biology & Biochemistry 32 (2000) 1475±1484

M.G., Fritsche, W., Hatakka, A., 1998. Oxidative decomposition of malonic acid as basis for the action of manganese peroxidase in the absence of hydrogen peroxide. FEBS Letters 434, 362±366. Jones, M.D., Durall, D.M., Tinker, P.B., 1990. Phosphorus relationships and production of extramatrical hyphae by two types of willow ectomycorrhizas at di€erent soil phosphorus levels. New Phytologist 115, 259±267. Jonsson, L., Dahlberg, A., Nilsson, M.-C., Zackrisson, O., KaÊreÂn, O., 1999a. Ectomycorrhizal fungal communities in late-successional Swedish boreal forests and composition following wild®re. Molecular Ecology 8, 205±215. Jonsson, T., Kokalj, S., Finlay, R., Erland, S., 1999b. Ectomycorrhizal community structure in a limed spruce forest. Mycological Research 103, 501±508. Kanunfre, C.C., Zancan, G.T., 1998. Physiology of exolaccase production by Thelephora terrestris. FEMS Microbiology Letters 161, 151±156. Kirk, T.K., Mozuch, M.D., Tien, M., 1985. Free hydroxyl radical is not involved in an important reaction of lignin degradation by Phanerochaete chrysosporium Burds. Biochemical Journal 226, 455±460. Kuhad, R.C., Singh, A., Eriksson, K.-E.L., 1990. Microorganisms and enzymes involved in the degradation of plant ®ber cell walls. In: Eriksson, K.-E.L. Biotechnology in the Pulp and Paper Industry. Advances in Biochemical Engineering/Biotechnology, vol. 57, 45±125. Lamar, R.T., Davis, M.W., Dietrich, D.M., Glaser, J.A., 1994. Treatment of a pentachlorophenol- and creosote-contaminated soil using the lignin-degrading fungus Phanerochaete sordida Ð a ®eld demonstration. Soil Biology & Biochemistry 26, 1603±1611. Lappin, H.M., Greaves, M.P., Slater, J.H., 1985. Degradation of the herbicide mecoprop (2-(2-methyl-4-chlorophenoxy) propionic acid) by a synergistic microbial community. Applied and Environmental Microbiology 49, 429±433. Leake, J.R., Read, D.J., 1997. Mycorrhizal fungi in terrestrial habitats. In: Wicklow, D., SoÈderstroÈm, B. (Eds.), The Mycota IV. Environmental and Microbial Relationships. Springer-Verlag, Berlin, pp. 281±301. Meharg, A.A., Cairney, J.W.G., 1999. Co-evolution of mycorrhizal symbionts and their hosts to metal contaminated environments. Advances in Ecological Research 30, 69±112. Meharg, A.A., Cairney, J.W.G., Maguire, N., 1997a. Mineralization of 2,4-dichlorophenol by ectomycorrhizal fungi in axenic culture and in symbiosis with pine. Chemosphere 34, 2495±2504. Meharg, A.A., Dennis, G.R., Cairney, J.W.G., 1997b. Biotransformation of 2,4,6-trinitrotoluene (TNT) by ectomycorrhizal basidiomycetes. Chemosphere 35, 513±521. Molina, R., Massicotte, H., Trappe, J.M., 1992. Speci®city phenomena in mycorrhizal symbiosis: community-ecological consequences and practical implications. In: Allen, M.F. (Ed.), Mycorrhizal Functioning. Chapman and Hall, London, pp. 357±423. Nicolotti, G., Egli, S., 1998. Soil contamination by crude oil: impact on the mycorrhizosphere and on revegetation potential of forest trees. Environmental Pollution 99, 37±43. Olsson, P.A., Chalot, M., BaÊaÊth, E., Finlay, R.D., SoÈderstroÈm, B., 1996. Ectomycorrhizal mycelia reduce bacterial activity in a sandy soil. FEMS Microbiology Ecology 21, 77±86. Paris, F., Botton, B., Lapeyrie, F., 1996. In vitro weathering of phlogopite by ectomycorrhizal fungi. II. E€ect of K+ and Mg2+ de®ciency and N sources on accumulation of oxalate and H+. Plant and Soil 179, 141±150. Rambelli, A., 1973. The rhizosphere of mycorrhizae. In: Marks, G.C., Kozlowski, T.T. (Eds.), Ectomycorrhizae, Their Ecology and Physiology. Academic Press, New York, pp. 299±349. Read, D.J., Boyd, R., 1986. Water relations of mycorrhizal fungi

and their host plants. In: Ayers, P., Boddy, L. (Eds.), Water, Fungi and Plants. Cambridge University Press, Cambridge, pp. 287±303. Rouillon, R., Poulain, C., Bastide, J., Coste, C.M., 1989. Degradation of the herbicide chlorpropham by some ectomycorrhizal fungi in pure culture. Agriculture, Ecosystems and Environment 28, 421±424. Rousseau, J.V.D., Sylvia, D.M., Fox, A.J., 1994. Contribution of ectomycorrhiza to the potential nutrient-absorbing surface of pine. New Phytologist 128, 639±644. Salt, D.A., Smith, R.D., Raskin, I., 1998. Phytoremediation. Annual Review of Plant Physiology and Plant Molecular Biology 49, 643±668. Sanderman, E., Loos, M.A., 1984. Enumeration of 2,4-D-degrading microorganisms in soils and crop plant rhizospheres using indicator media: high populations associated with sugarcane (Saccharum ocinarum ). Chemosphere 13, 1073±1084. Sarand, I., Timonen, S., Koivula, T., Peltola, R., Haahtela, K., Sen, R., Romantschuk, M., 1999. Tolerance and biodegradation of mtoluate by Scots pine, a mycorrhizal fungus and ¯uorescent pseudomonads individually and under associative conditions. Journal of Applied Microbiology 86, 817±826. Sarand, I., Timonen, S., Nurmiaho-Lassila, E.-L., Koivila, T., Haahtela, K., Romantschuk, M., Sen, R., 1998. Microbial bio®lms and catabolic plasmid harbouring degradative ¯uorescent pseudomonads in Scots pine mycorrhizospheres developed on petroleum contaminated soil. FEMS Microbiology Ecology 27, 115± 126. Sawyer, N.A., Chambers, S.M., Cairney, J.W.G., 1999. Molecular investigation of genetic distribution and genetic variation of Cortinarius rotundisporus in eastern Australian sclerophyll forests. New Phytologist 142, 561±568. Selosse, M.-A., Martin, F., Bouchard, D., Le Tacon, F., 1999. Structure and dynamics of experimentally introduced and naturally occurring Laccaria sp discrete genotypes in a Douglas ®r plantation. Applied and Environmental Microbiology 65, 2006± 2014. Singleton, I., 1994. Microbial metabolism of xenobiotics: fundamental and applied research. Journal of Chemical Technology and Biotechnology 59, 9±23. Smith, S.E., Read, D.J., 1997. Mycorrhizal Symbiosis. Academic Press, London. Sun, Y.-P., Unestam, T., Lucas, S.D., Johanson, K.J., Kenne, L., Finlay, R., 1999. Exudation-reabsorption in a mycorrhizal fungus, the dynamic interface for interaction with soil and soil microorganisms. Mycorrhiza 9, 137±144. Thomas, A.O., Lester, J.N., 1994. Reclamation of disused gaswork sites: new solutions to an old problem. Science of the Total Environment 152, 239±260. Timonen, S., Sen, R., 1998. Heterogeneity of fungal and plant enzyme expression in intact Scots pine-Suillus bovinus and Paxillus involutus mycorrhizospheres developed in natural forest humus. New Phytologist 138, 355±366. Trojanowski, J., Haider, K., HuÈttermann, A., 1984. Decomposition of 14C-labelled lignin, holocellulose and lignocellulose by mycorrhizal fungi. Archives of Microbiology 139, 202±206. Villeneuve, N.F., Le Tacon, F., Bouchard, D., 1991. Survival of inoculated Laccaria bicolor in competition with native ectomycorrhizal fungi and e€ects on the growth of outplanted Douglas-®r seedlings. Plant and Soil 135, 95±107. Walton, B.T., Anderson, T.A., 1990. Microbial degradation of trichloroethylene in the rhizosphere: potential application to biological remediation of waste sites. Applied and Environmental Microbiology 56, 1012±1016.