K.Sivasithamparam, K.W.Dixon & R.L.Barrett (eds) 2002. Microorganisms in Plant Conservation and Biodiversity. pp. 105–150. © Kluwer Academic Publishers. Chapter 5 ECTOMYCORRHIZAS IN PLANT COMMUNITIES Mark C. Brundrett CSIRO Forestry and Forest Products, CSIRO Centre for Mediterranean Agricultural Research, Private Bag No 5, Wembley, 6913, Western Australia; Soil Science and Plant Nutrition, Faculty of Natural and Agricultural Sciences, The University of Western Australia, Crawley 6009, Western Australia (current address) and Science Directorate, Kings Park and Botanic Garden, Botanic Gardens and Parks Authority, West Perth 6005, Western Australia (correspondence). John W.G. Cairney Mycorrhiza Research Group, Centre for Horticulture and Plant Sciences, Parramatta Campus, University of Western Sydney, Locked Bag 1797, Penrith South DC, 1797, New South Wales, Australia. 1. Introduction 1.1. Associations Ectomycorrhizal associations (abbreviated as ECM) are sometimes called ectotrophic associations or sheathing mycorrhizas. They are mutualistic associations between higher fungi and Gymnosperm or Angiosperm plants belonging to the families listed in Table 1. These associations consist of mycorrhizal roots and fungal storage or reproductive structures that are interconnected by soil-borne mycelia (Figure 1). Ectomycorrhizal associations are formed predominantly on the fine root tips of the host. These ECM roots are defined by the presence of a mantle, consisting of interwoven hyphae on the root surface, and a Hartig net, which is a labyrinth of highly branched hyphae between cells of the root epidermis or cortex. These structures are not always both well developed in the same association. These roots and their associated fungal hyphae typically are most abundant in topsoil layers containing humus and are thought to make a substantial contribution to soil biomass and nutrient cycling in many ecosystems (Section 3.1). Detailed descriptions of the structure and development of ECM are available elsewhere (e.g. Kottke and Oberwinkler 1986; Massicotte et al. 1987). 106 Microorganisms in Plant Conservation and Biodiversity A . Example of ECM short roots (arrows) of birch (Betula alleghaniensis). The mycorrhizal short roots are much thicker than other laterals of the same order (grid = 1 mm). B . ECM short roots (S) of Eucalyptus globulus with a Cenococcum-like mycorrhiza that has relatively thick black radiating external hyphae (arrow). (Magnification = approx. 40x Photo courtesy of Nick Malajczuk). C . Ectomycorrhizal association with highly branched short roots with many root tips (arrows). This Pinus radiata and Amanita muscaria association was synthesised under sterile conditions. (Magnification approx. 24x, Photo courtesy of Nick Malajczuk, Randy Molina and Jim Trappe). D . Labyrinthine Hartig net hyphae (arrows) on elongated epidermal cells (E) of a Populus tremuloides ECM root. The mantle (M), cortex (C) and endodermis (En) are also visible. This is a cleared and stained root hand section. (Magnification 540x) Figure 1. Ectomycorrhizal roots and microscopic structures. 1.2. Host plants Trees with ECM associations typically are dominant in coniferous forests in boreal or alpine regions, but are also important in some temperate deciduous forest, tropical forest, as well as savannah and mediterranean plant communities (Meyer 1973; Högberg 1986; Brundrett 1991). Plant families reported to have ECM are listed in Table 1. This table excludes Ectomycorrhizas in plant communities 107 families with arbutoid or monotropoid ECM associations and those with atypical ECM associations, such as Australian herbaceous plants in the families Goodeniaceae, Asteraceae and Stylidiaceae (Kope and Warcup 1986; Brundrett 1999b). The majority of ECM hosts are trees, or shrubs (Table 1), but associations are formed by a few herbaceous plants, including Kobresia, Polygonum and Cassiope species found in arctic or alpine regions (Kohn and Stasovski 1990; Massicotte et al. 1998). 1.3. Fungi The reproductive structures of ECM fungi include epigeous fungi (mushrooms, puffballs, coral fungi, etc.) and subterranean structures (hypogeous fungi which are called truffles or truffle-like fungi). Most epigeous fungi have a hymenium consisting of gills, pores, teeth, etc. which actively releases spores, but the hypogeous fungi and puffballs have sequestrate fruit bodies which enclose their spores. The majority of ECM fungi are Basidiomycetes, but there also are a number of Ascomycetes and a handful of Zygomycetes (Molina et al. 1992). Most identification guides for larger fungi provide information about their probable host plants from field observations. Lists of known Australian ECM fungal genera and those which associate with Eucalyptus species are provided on the Web (Brundrett and Bougher 1999). Detailed descriptions of the process required to collect, document, store and identify fungal specimens are provided in manuals (e.g. Largent 1986; Brundrett et al. 1996c). However, accurate fungal identification is a time consuming process that requires much expertise. Fungal identification is most difficult in tropical regions and the southern hemisphere, where many species have yet to be described or illustrated in identification guides. In particular, the hypogeous ECM fungi have been neglected in many regions (Bougher and Lebel 2001). Due to these taxonomic difficulties, it is probable that many lists of fungi contain errors. Consequently, it is imperative that voucher specimens of all fungi referred to in publications are lodged with an internationally recognised herbarium, and these specimens contain material of sufficient quality and quantity to allow future taxonomic and DNA-based studies. 2. Fungal biology 2.1. Distribution and diversity Information on the identity and relative abundance of ECM fungi present in a particular habitat is required to understand the relationship between taxonomic and functional diversity (Section 4.1). Approximately 5500 species of ECM fungi have been listed world-wide (Molina et al. 1992). However, this list would significantly underestimate the diversity of these fungi, as the fungal flora in tropical and southern regions is poorly known. For example, it has been estimated that there could be as many as 6500 species of ECM fungi in Australia alone, but only about 700 species from this region have been named so far (Bougher 1995; Brundrett and Bougher 1999). Most estimates of diversity are based on Microorganisms in Plant Conservation and Biodiversity 108 surveys of obvious epigeous fruiting bodies, which exclude hypogeous and resupinate fungi. Table 1. Ectomycorrhizal plants. Family Gymnosperms Gnetaceae Pinaceae Angiosperms Monocots Cyperaceae1 Fagales Betulaceae Casuarinaceae Corylaceae Fagaceae Juglandaceae Myricaceae Nothofagaceae Legumes Caesalpiniaceae3 Growth form Genera climber tree Gnetum Abies, Cathaya, Cedrus, Keteleeria, Larix, Picea, Pinus, Pseudolarix, Pseudotsuga, Tsuga herb Kobresia tree/shrub tree/shrub shrub tree tree shrub Alnus, Betula, Carpinus, Ostrya, Ostryopsis Allocasuarina, Casuarina2 Corylus Castanea, Castanopsis, Fagus, Quercus Carya, Castanopsis, Engelhardtia Myrica, Comptonia Nothofagus tree Anthonotha, Afzelia, Berlinia, Brachystegia, Eperua, Gilbertiodendron, Gleditsia, Intsia, Isoberlinia, Julbernardia, Microberlinia, Monopetalanthus, Tetraberlinia Gastrolobium, Gompholobium, Jacksonia, Mirbelia, Oxylobium, Pericopsis, etc. Acacia2 Fabaceae3 shrub Mimosaceae3 Other Dicots Cistaceae Dipterocarpaceae shrub/tree Helianthemum, Cistus, Tuberaria Anisoptera, Dipterocarpus, Hopea, Marquesia, Monotes, Shorea, Vateria Cassiope, Arbutus Ericaceae shrub Marquesia, Uapaca Euphorbiaceae3 tree Owenia Meliaceae tree Allosyncarpia, Agonis, Angophora, Baeckea, Myrtaceae3 shrub/tree Eucalyptus, Leptospermum, Melaleuca, etc. Neea, Pisonia Nyctaginaceae tree Polygonum Polygonaceae 1 herb Pomaderris, Trymalium Rhamnaceae3 shrub Dryas Rosaceae3 shrub Populus, Salix Salicaceae tree Tilia Tiliaceae tree Data are from Brundrett et al. (1996c). Notes: 1. Families with many non-mycorrhizal plants. 2. Acacia and Casuarina often have VAM. 3. Families with many VAM plants. Families requiring further study or have atypical associations are excluded (see Brundrett 1999 for a more complete list). shrub tree Ectomycorrhizas in plant communities 109 Ectomycorrhizal fungi are normally identified by observations of fruiting under their putative hosts, but the relationship between fruit bodies and the activity of mycelia in soils has usually not been established. The recognition of fungi by ECM morphology (colour, texture, structure, size, branching, etc.), has provided a powerful tool for the identification of fungi in individual root tips (e.g. Agerer 1995; Bradbury et al. 1998; Hagerman et al. 1999; Massicotte et al. 1999). Lipid profiles can also be used to identify ECM fungi in soils (Olsson 1999). Methods based on DNA are now often employed to identify ECM roots and provide a much more accurate picture of below-ground fungal activity than observations of fruit bodies (e.g. Dahlberg and Stenlid 1995; Horton and Bruns 1998; Jonsson et al. 1999a,b). A list of molecular sequence data that can help to identify ECM fungi is provided by Bruns et al. (1998). Molecular studies of fungi inhabiting ECM root tips in the field have shown that up to 60% of ECM roots are inhabited by fungi not observed to produce obvious epigeous fruiting bodies (e.g. Gardes and Bruns 1996; Jonsson et al. 1999a,b). These cryptic fungi would likely include those with hypogeous sequestrate (truffle-like) fruiting bodies (Castellano and Bougher 1994), or inconspicuous, epigeous, resupinate fruiting bodies (Erland and Taylor 1999), largely ignored in previous diversity surveys. Fungi such as Cenococcum, which do not produce macroscopic fruit bodies, also appear to be widespread. However, the observed discrepancies between the fungi which form mycorrhizas and those which fruit at a particular site seem to be due to the fact that many fungi reproduce very sporadically. Most genera of ECM fungi are reported to be widely distributed throughout the world, but there are also genera restricted to certain regions. Individual species of ECM fungi are often restricted to particular geographic regions. When considering functional aspects of mycorrhizal associations we should consider isolates of fungi rather than species or genera, because considerable intraspecific physiological variation is known to exist in ECM fungi (Trappe 1977; Brundrett 1991; Cairney 1999). Thus information about the soil and climatic conditions and host species present where fungi occur may be as important as their accurate identification. Future taxonomic studies are likely to reveal much finer differences between taxa of fungi, than are used in current classification schemes, especially for the neglected floras of the tropics and southern hemisphere. Molecular investigations are also revealing that taxa previously regarded as conspecific are complexes of several species. A good example is the cosmopolitan species Pisolithus tinctorius, for which a number of putative species have been identified using restriction fragment polymorphism (RFLP) and sequence analyses of the rDNA internal transcribed spacer (ITS) region (Anderson et al. 1998; Martin et al. 1998; Chambers and Cairney 1999; Sims et al. 1999). These approaches are likely to result in taxonomic revisions of many other genera of ECM fungi and provide us with a greater understanding of correlation between the taxonomy and physiological attributes of fungi (Section 1.3). 110 Microorganisms in Plant Conservation and Biodiversity 2.2. Lifecycles and inoculum There are a number of distinct stages in the life cycle of a mycorrhizal association (Table 2). These stages often occur at particular times of the year. Mycorrhizal exchange is a short-lived process dependent on root growth (Downes et al. 1992; Cairney and Alexander 1992a,b; Massicotte et al. 1987) – which in turn is regulated by environmental conditions and host plant phenology. Fungal fruiting, and thus the availability of spore inoculum, is also regulated by climatic factors and fungal phenology. It is important to think of ECM associations as dynamic processes as fungi would utilise mycelia and spores to move through soil while competing with other fungi to claim new roots. Thus, the size and shape of individual fungi is constantly changing with time, due to their foraging behaviour (Section 3.1), interactions with other soil organisms (Section 2.3) and environmental factors (Section 3D). These dynamic processes may, in part at least, explain discrepancies between the fungi fruiting above ground and those forming mycorrhizas in a particular location (Section 2.1). Propagules of ECM fungi include individual hyphae, strands (aggregations of hyphae), spores, sclerotia and probably also mycorrhizal roots (Ogawa 1985; Fries 1987; Ba et al. 1991; Miller et al. 1994; Torres and Honrubia 1997). Most ECM fungi do not produce conidia (Hutchinson 1989). Boreal forest soil and leaf litter often contains spores capable of initiating mycorrhizas (Amaranthus and Perry 1987; Parke et al. 1983b). Localised patterns of ECM fungus proliferation depend on the production of hyphae or strands by a particular endophyte (Ogawa 1985; Agerer 1995; Unestam and Sun 1995). Mycelia of some ECM fungi are thought to require attachment to a living host root to initiate new mycorrhizas (Fleming et al. 1984). Ectomycorrhizal short roots live for months (Majdi and Nylund 1996; Rygiewicz et al. 1997) and are often protected by a thick covering of mantle hyphae, suggesting that they may be important fungus survival structures. However, it is not known how long ECM fungi in these roots can survive after detachment from the host. The implications of ECM fungus dispersal and survival are considered in sections 2.3 and 2.4 below. 2.3. Consumption and dispersal Ectomycorrhizal fungus structures are a major structural component of certain soils, and thus are an important food source for many soil organisms (Table 3). Hyphal grazing by soil organisms can reduce mycorrhiza formation and nutrient translocation by hyphae in soils (Hiol et al. 1995; Setälä 1995). Soil organisms which ingest, inhabit, or associate with hyphae or sporophores of ECM fungi include members of most soil trophic levels (Table 3). Dense mats of ECM roots and mycelia in forest soils can have substantially higher populations of microbes and micro-arthropods than other areas (Cromack et al. 1988; Griffiths et al. 1991). Ectomycorrhizas in plant communities 111 Table 2. The life cycle of a mycorrhizal association. Stage • 1. Fungal propagules [spores, hyphae, old roots, etc.] • • • • 2. Active soil hyphae • [mineral nutrient • acquisition and spread] • • Root growth • [requires young roots] • Hyphal • proliferation on • root surface • Hyphal penetration • between root cells • to form an • exchange site • [Hartig net] 3. 4. 5. 6. 7. Active exchange processes Senescence of structures in roots • • • • • • • • 8. Propagule formation [the cycle continues] 9. Death and decomposition • • • • • Note: earlier events may continue Components and factors influencing their survival or functioning disturbance, predation adverse soil conditions dispersal mechanisms activation — fungi may respond to: 1. environmental conditions 2. time intervals (dormancy, quiescence) 3. presence of roots or other organisms microhabitat preferences? limited saprobic potential? attraction to young host roots? tropic responses? regulated by phenology and environmental factors production of soluble or volatile signals recognition of potential hosts? hyphae respond by changing morphology mantle structures controlled by fungus avoidance or tolerance of host defences recognition by host may result in cell enlargement pronounced fungus morphology changes host cellular responses (synthesis of cytoplasm, secondary metabolites, etc.) hyphal penetration limited by host cell properties transfer of minerals obtained by soil mycelium to host. simultaneous transfer of food to fungus limited in duration influence of host, fungus or environment? disorganisation of cytoplasm in exchange site hyphae fungal resources withdrawn for storage by mantle hyphae transfer to external mycelium, strands or rhizomorphs sexual spores produced by fruit bodies, strands and sclerotia mycelial networks in soil old mycorrhizal roots? outer root layers (fungus habitat) lost due to: 1. root death or secondary growth 2. parasitism or consumption 3. soil disturbance in parallel with later events. Dispersal of fungal propagules is required for colonising new habitats, increasing fungal diversity, changing fungal population structure, or introducing new genes to existing fungi. Most localised 112 Microorganisms in Plant Conservation and Biodiversity spread is by mycelial growth through the soil, resulting in discrete patches of soil occupied by the hyphal networks of individual fungi, that can be distinguished from others of their species using genetic or DNAbased methods (de la Bastide et al. 1994; Dahlberg and Stenlid 1995). Many fungi forming ECM associations have large fruiting structures (mushrooms) that produce abundant wind-borne spores, but survival and dispersal of these spores may be limited. Certain ECM fungi produce sclerotia which probably are much more resilient than other propagules (Miller et al. 1994). Fungi with hypogeous fruiting bodies are often excavated and consumed by small mammals or marsupials and thus spread to new locations (Table 3). Spores of ECM fungi contained in animal faeces are a viable source of inoculum (Claridge et al. 1992; Cázares and Trappe 1994; Reddell et al. 1997). 2.4. Disturbance Severe disturbance includes situations where vegetation has been lost and topsoil has been removed or mechanically disrupted, or where plants are introduced to new substrates resulting from mining, glaciation, or volcanic activity. Soil disturbance can result in temperature extremes, anaerobic conditions, loss of organic matter, loss of nutrients, structural changes and loss of biological components (Abdul-Kareem and McRae 1984; Danielson 1985). There is a high degree of spatial variability in ECM fungus inoculum in Australian natural habitats (Brundrett and Abbott 1995; Brundrett et al. 1996a), but this variability is even larger in disturbed habitats, where large gaps between patches containing inoculum would prevent many seedlings from encountering these fungi (Brundrett et al. 1996b). Forestry activities can also result in reductions in ECM fungus inoculum due to the absence of host plants and soil degradation (Amaranthus and Perry 1987; Parke et al. 1983a; Harvey et al. 1997; Perry et al. 1987; Visser et al. 1998; Hagerman et al. 1999). After mechanical disturbance of soils, surviving ECM fungal inoculum may be concentrated in localised soil pockets high in organic materials (Christy et al. 1982; Parke et al. 1983b; McAfee and Fortin 1989). Other forms of severe disturbance known to adversely affect ECM fungi include erosion and fires (Table 3). Mycorrhizal inoculum is often limited in recently disturbed habitats (Danielson 1985; Malajczuk et al. 1994; Brundrett et al. 1996b; Reddell et al. 1999). Propagules expected to survive in disturbed soils are thought to include mycorrhizal root fragments, hyphae within organic matter, segments of rhizomorphs, sclerotia and perhaps spores and root pieces (Ba et al. 1991; Brundrett 1991). The networks of fungal hyphae which are the main propagules of ECM fungi would be highly susceptible to disturbance and other more resilient propagules would decline in the absence of host roots (Figure 2). Reductions in fungal diversity could result because fungi have a limited capacity to adapt to major changes in environmental conditions (Table 4). Fungus specificity (Section 3.3) may also prevent mycorrhiza formation if surviving fungi are not compatible with new host plants (e.g. fungi from Eucalyptus may not form Ectomycorrhizas in plant communities 113 mycorrhizas if Acacia or Melaleuca are dominant after disturbance). The time required for fungi to grow from residual inoculum or re-colonise sites by dispersal, will determine the rate of recovery of fungal diversity. Table 3. Organisms that interact with ECM fungi. Organism Bacteria and actinomycetes Protoctistan animals (Amoebae) Higher fungi Nematodes Arthropods (mites, springtails, beetles, etc.) Larger animals squirrels and other small mammals deer, goats marsupials Type of References association 1, 2 Garbaye 1994; Olsson and Wallander 1998; Mogge et al. 2000 1, 2 Cromack et al. 1988; Griffiths et al. 1991; Ingham and Massicotte 1994; Jentschke et al. 1995 1, 2, 5 Summerbell 1989 1, 2, 3, 4 Sutherland and Fortin 1968; Cromack et al. 1988 1, 3, 4, 6 Cromack et al. 1988; Setälä 1995; Lawrence and Milner 1996 Maser and Maser 1988; Cázares et al. 1999 5, 6 Cázares and Trappe 1994 5, 6 Claridge et al. 1996; Johnson 1995; 1996; McIlwee and Johnson 1998; Reddell et al. 1997 humans 5, 6 e.g. Arora 1991; Kalotas 1996; Hosford et al. 1997 Categories: 1 = associated with mycorrhizas; 2 = associated with mycelia; 3 = feed o n hyphae; 4 = feed on mycorrhizal roots; 5 = feed on epigeous fruit bodies; 6 = feed o n hypogeous fruit bodies. 5, 6 Spore dispersal by the wind and mycophagous animals (Table 3) are considered important in the colonisation of new habitats by ECM fungi (Cázares and Trappe 1994; Johnson 1995; Brundrett et al. 1996b). The effectiveness of these natural vectors will depend on the proximity of disturbed sites to habitats containing suitable fungi (and their associated animals) as well as the phenology of fruiting of fungi. It is not known if these fungi are more or less readily dispersed than their host plants (Figure 2). Unfortunately, there is insufficient information about the biology of mycorrhizal fungi to make robust predictions about the capacity of particular strains of fungi to survive disturbance, or to adapt to changes in soil conditions following disturbance. Fungal communities in disturbed habitats are considered further in Section 4.1. 114 Microorganisms in Plant Conservation and Biodiversity Figure 2. Probable impacts of disturbance, time without host plants and habitat changes on ECM fungi. (Arrow thickness is proportional to propagule survival.) 3. Mycorrhizal plants 3.1. Benefits to plants Ectomycorrhizal associations are assumed to have key roles in nutrient cycling processes in ecosystems where their hosts are dominant. This assumption is based on the large biomass of their fruit bodies which appear at certain times (Fogel and Hunt 1979; Vogt et al. 1982) and the pervasiveness of mycelium assumed to belong to ECM fungi in soils. Further evidence is provided by measurements of substantial carbon transfer from host plants to ECM fungi (Rygiewicz and Andersen 1994; Markkola et al. 1995; Setälä et al. 1999) and between interconnected plants (Simard et al. 1997). The mycelia of ECM fungi also are a major structural component of soils (Table 5). Glasshouse experiments have provided many demonstrations of substantial benefits from inoculation with ECM fungi, due to growth responses resulting from enhanced Ectomycorrhizas in plant communities 115 nutrient uptake, improved disease resistance, etc. (Table 5). Unfortunately, there have been few attempts to measure these parameters in natural ecosystems and it is dangerous to extrapolate results from highly simplified experimental systems to the real world. Table 4 . Soil factors influencing the occurrence, distribution or effectiveness of ECM fungi. Soil condition Soil organic matter Forestry activities Erosion Waterlogging Drought Salinity Soil pH and aluminium Toxic levels of metals Pesticides or organic pollutants Air pollution (O3, N, S) Elevated CO2 Soil fertility (P) High soil N Intense fire Low or high temperatures References de Vries et al. 1995; Baar and Kuyper 1988 Parsons et al. 1994; Harvey et al. 1997; Zhou and Sharik 1997; Visser et al. 1998; Hagerman et al. 1999 Amaranthus and Trappe 1993 Slankis 1974; Bougher and Malajczuk 1990; Khan 1993; Khan and Belik 1995 Coleman et al. 1989; Boyle and Hellenbrand 1991; Osonubi et al. 1991 Dixon et al. 1993 Aggangan et al. 1996; Thomson et al. 1996; Schier and McQuattie 1996; Qian et al. 1998 Hartley et al. 1997; Leyval et al. 1997 Sidhu and Chakravarty 1990; Nicolotti and Egli 1998; Aleksandrowicz-Trzcinska and Grzywacz 1997 Termorshuizen and Schaffers 1987; Markkola et al. 1995; Brandrud et al. 1998 Godbold and Berntson 1997; Berntson et al. 1997; Runion et al. 1997; Walker et al. 1999a Arnebrant and Söderstrom 1992; Gehring and Whitham 1994; Walker et al. 1999a Tétreault et al. 1978; Taylor and Alexander 1989; Haug et al. 1992; Majdi and Nylund 1996 Torres and Honrubia 1997; Horton et al. 1998; Baar et al. 1999 Parke et al. 1983a; Cline et al. 1987; McInnes and Chilvers 1994; Soulas et al. 1997 The “foraging behaviour” of ECM fungus mycelia results in proliferation within nutrient rich patches of soil, lowering nutrient concentrations in these zones (Bending and Read 1995). Different parts of the mycelial systems of ECM fungi exhibit different physiological capabilities and structural properties (Unestam and Sun 1995; Cairney and Burke 1996). Only a fraction of the hyphae of an ECM fungus is considered to be capable of nutrient uptake at any one time and this proportion decreases with association age (Taylor and Peterson 1998). Mycelial systems produced by ECM fungi in soils are considered to play a key role in nutrient cycling in many ecosystems and function as the primary soil-plant interface for their hosts, which include many important 116 Microorganisms in Plant Conservation and Biodiversity forest trees. Unfortunately, there have been relatively few attempts to study ECM fungus systems in-situ. Some knowledge has come from observations of large (≤ 1 m wide) patches dominated by the mycelium of some ECM fungi, where soil physical and chemical properties are altered (Griffiths et al. 1994, 1996; Unestam and Sun 1995). It is considered that changes to soil properties (higher ion concentrations, oxalate accumulation, etc.) in these “hyphal mats” result in increased nutrient availability due to accelerated weathering of soil minerals (Griffiths et al. 1994; Paris et al. 1995). Hyphae of ECM fungi have also been implicated in the weathering of rock fragments in soils (Jongmans et al. 1997; Landeweert et al. 2001). The role of ECM fungi in nutrient cycling depends on their ability to acquire the mineral nutrients required by host plants from inorganic and organic sources in soils. It is generally assumed that ECM fungi have a greater capacity to acquire organic forms of nutrients than AM fungi (Marschner 1995; Smith and Read 1997). Ectomycorrhizal roots normally are more abundant in topsoil layers containing humus, than in underlying layers of mineral soil (Meyer 1973; Harvey et al. 1978; 1997). Evidence for the utilisation of organic materials by ECM fungi has been provided by production of enzymes capable of breaking down organic N sources in experimental systems (Table 5) and 15N studies of ECM plants and fungi (Michelsen et al. 1998). Some ECM fungi specialise in the breakdown of organic compounds in animal wastes (Sagara 1995; Yamanaka 1999). Observations of substrate utilisation and isotopic composition of fungi have shown that ECM fungi generally have a much lower capacity to degrade complex substrates such as cellulose, lignin or phenolics than saprophytic fungi (Dighton et al. 1987; Bending and Read 1997; Kohzu et al. 1999). However, measurements of 15N content of fruit bodies by Gebauer and Taylor (1999) suggested that some ECM fungi primarily utilised organic N sources from humus, while others depend on inorganic N from the soil. Organic N sources have been shown to be important to plants with ECM in arctic tundra, Australian eucalypt forests and boreal forests (Kielland 1994; Turnbull et al. 1995). The impact of different nutrient sources on competition between plants is considered in Section 4.2. In situ studies of litter decomposition have shown that hyphae considered to belong to ECM fungi were only present in the latter stages of this complex process and most of the work is done by other types of soil microbes and animals (Ponge 1991). The presence of ECM fungi may increase, or reduce the rate of breakdown of soil organic matter (Gadgil and Gadgil 1975; Dighton et al. 1987). Even if some ECM fungi have a role in organic matter breakdown, this would be less important than their role in coupling plants into the soil food web. Lindahl et al. (1999) studied interactions between mycelia of several ECM fungi and a wood rotting fungus (Hypholoma) in a microcosm experiment. They observed antagonistic interaction by several ECM fungi on Hypholoma, resulting in substantial transfer of 32P to the mycorrhizal fungus. We still have much to learn about how ECM fungi acquire nutrients directly from Ectomycorrhizas in plant communities 117 organic sources or indirectly from other soil organisms responsible for nutrient cycling and how these processes are affected by environmental factors. Ectomycorrhizal fungi are the final step in the soil nutrient cycling process over a large portion of the earth’s surface, a role that is essential for ecosystem sustainability. Table 5. Suggested roles of ectomycorrhizal fungi in natural and managed ecosystems. A. Benefits to plants 1 . Increased plant nutrient supply by extending the soil volume accessible to root systems (Marschner 1995). 2 . Increased plant nutrient supply by acquiring organic nutrient forms that are not otherwise available to plants (Turnbull et al. 1995; Chalot and Brun 1998; Näsholm et al. 1998). 3 . Antagonism of parasitic organisms (Bhat et al. 1997; Morin et al. 1999). 4 . Non-nutritional benefits to plants due to changes in water relations, phytohormone levels, carbon assimilation, secondary metabolite production (Brundrett 1991; Beyeler and Heyser 1997; Smith and Read 1997). 5 . Carbon transfer through ECM fungus mycelia connecting plants (Simard et al. 1997). B. Other roles in ecosystems 6 . Soil hyphae could prevent nutrient losses, especially at times when roots are inactive (Lussenhop and Fogel 1999). 7 . Sporocarps are important food sources for larger animals (Claridge et al. 1996; McIlwee and Johnson 1998). 8 . Sporocarps and mycelia are important as food sources and habitats for invertebrates (Ingham and Massicotte 1994; Lawrence and Milner 1996). 9 . Some ECM fungi help detoxify allelopathic compounds in soil (Hanson and Dixon 1987). 10. Some ECM fungi recycle nutrients in animal wastes (Sagara 1995; Yamanaka 1999). 11. ECM fungi may detoxify allelopathic chemicals in soils (Hanson and Dixon 1987). 12. Hyphae may influence soil structure and soil chemistry (Griffiths et al. 1991). 13. They contribute to carbon storage in soil by altering the quality and quantity of soil organic matter (Rygiewicz and Andersen 1994; Setälä et al. 1999). 14. Hyphae may transport carbon from plant roots to help support other soil organisms involved in nutrient cycling processes. C. Values to people 15. Fruit bodies of ECM fungi are economically and nutritionally important as human food resources (Arora 1991; Kalotas 1996). 16. We also use them as medicines and natural dyes (Arora 1991; Morgan 1995). 17. Fungi have aesthetic values and are an important part of the culture, folklore and appreciation of nature by many people (Findlay 1989; Morgan 1995). 18. Fungal diversity is a valuable bio-indicator of environmental quality. 19. Ectomycorrhizas are required for growth of many trees used in plantation forestry. 20. Fungi can help plants grow in soils we have polluted with metals or organic chemicals (Hartley et al. 1997; Meharg and Cairney 2000b). 118 Microorganisms in Plant Conservation and Biodiversity 3.2. Mycorrhizal dependency Different categories of mycorrhizal dependency have been defined for plants with AM associations (Chapter 4, Section 3.2), but it is generally assumed that all plants with ECM are obligately mycorrhizal (unable to survive to reproductive maturity without fungi). Experiments demonstrating substantial growth responses to ECM fungus inoculation are too numerous to list here and involve many of the plant genera listed in Table 1. However, these experiments normally use relatively infertile substrates that are initially devoid of ECM fungi and favourable environmental conditions. Demonstrations of growth responses due to mycorrhizal inoculation in the field have been less common, probably because mycorrhizal fungi are already present, soils may be more fertile and environmental conditions are not always suitable for fungal activity (Castellano 1994; Jackson et al. 1995; Brundrett 2000). Practical uses of ECM fungi are considered in section 5. There normally is a strong positive correlation between a plant’s dependency on mycorrhizas and the degree of mycorrhizal formation in its roots (Janos 1980; Brundrett 1991). Reports of ECM host plants in natural habitats without these structures on most of their fine root tips are rare. However, trees growing in flooded soils (e.g. Salix, Populus and Melaleuca) can have low levels of ECM, relative to trees of the same species in drier soils (Lodge 1989; Khan and Belik 1995). Trees with ECM even occur naturally in extremely fertile soils, such as Pisonia on coral islands where nesting birds provide massive fertiliser inputs (Ashford and Allaway 1985). In this case mycorrhizas appear to be involved in acquiring transiently-available nitrogenous products of uric acid degradation prior to leaching from the coral cay soils (Sharples and Cairney 1997). Perhaps the best evidence that hosts have an obligate requirement for mycorrhizas come from plantation forestry, where the failure of trees such as pines to establish without mycorrhizal inoculum has been noted when they are first planted in exotic locations (Trappe 1977; Smith and Read 1997). Further evidence comes from the nature of the root systems of host trees such as conifers with thick, slow-growing roots without long root hairs that contrast starkly with the fine root systems of plants that are able to grow well without mycorrhizas (Brundrett 1991; Marschner 1995). 3.3. Specificity Three different categories of host-fungus specificity were defined by Molina et al. (1992), who provide lists of fungi considered to belong in each category. Narrow host range fungi are only known to associate with one genus of host plants, intermediate host range fungi associate with different species of hosts within a single plant family or group such as the Gymnosperms and broad host range fungi form mycorrhizas with plants from unrelated families. Most knowledge about the host specificity of fungi is based on observations of fruit bodies under trees, so it should be assumed that lists of fungal associates for plant taxa contain some errors. Ectomycorrhizas in plant communities 119 For example, the fungus Boletinellus merulioides and the tree Fraxinus appear in lists as ECM associates, but this tree only has AM in its roots and this fungus forms a mutualistic relationship with a subterranean aphid (Brundrett and Kendrick 1987). Further evidence of specificity is provided by glasshouse and sterile culture synthesis experiments which confirm that fungi can form normal looking mycorrhizas with some host plants but not others (e.g. Malajczuk et al. 1982; Burgess et al. 1993). However, some host-fungus combinations which form mycorrhizas in soil are incapable of doing so in sterile environments and vice versa (Malajczuk et al. 1982; Ba et al. 1994). Baiting experiments, where different ECM fungi were detected in the same soil using different host plants, have also demonstrated fungus specificity (Jones et al. 1997; Massicotte et al. 1999). Lists of ECM host-fungus associations are based on observations of fungal fruiting under certain plants backed up by the knowledge gained from synthesis experiments. Consequently, we must expect such lists to contain some errors. Although some ECM fungi, including Cenococcum geophilum (LoBuglio 1999) and Hebeloma cristuliniforme (Marmeisse et al. 1999) have been reported to form ECM with a diverse array of host genera belonging to different plant families, many have a much narrower host range (Molina et al. 1992). These narrow range fungi commonly show specificity at the host genus level with, for example, ca. 250 taxa thought to form ECM only with Douglas fir (Pseudotsuga) in North America (Molina et al. 1992) and a much larger number are likely to be restricted to Eucalyptus in Australia (Bougher 1995). We must be mindful, however, that host ranges of the majority of ECM fungi have been inferred from observations of co-occurrence of sporocarps and hosts in the field (Molina et al. 1992). In many cases it is impossible to separate effects of geographical distribution from true host specificity. Some reports of fungi with a single host may result from our limited knowledge of fungal distribution patterns in many regions. Intermediate host range fungi appear to be most common (Molina et al. 1992; Horton and Bruns 1998). This notwithstanding, individual trees in the field are normally colonised by both narrow and broad host range mycobionts and this may have important influences on competitive interactions between tree species (Section 4.2). The number of species of fungi in the broad host range category may well decrease with future taxonomic studies, as widely distributed fungi are found to comprise a number of similar looking species. A good example of this is the species Pisolithus tinctorius – reported to be one of the most common ECM fungi in the world, with a wide range of reported hosts and habitats. However, recent molecular, and biochemical evidence suggests that this taxon consists of a number of species with more restricted host and habitat preferences (Section 2.1). Perhaps the best evidence that most ECM fungi are relatively specific comes from the low diversity of fungi which occur in plantations of pines and eucalypts grown in exotic locations (Dunstan et al. 1998; Brundrett and Bougher 1999). In many countries only a few genera have been 120 Microorganisms in Plant Conservation and Biodiversity reported to occur with these hosts, but there are many species found under indigenous tree species. The number of species of fungi fruiting in Australian Eucalyptus plantations increases with time in Australia, but remains low, even after many years, in most exotic locations (Lu et al. 1999). Newton and Haigh (1998) found that diversity of ECM fungi associated with particular hosts was positively correlated with the area of the UK occupied by these hosts plants. However, exotic trees introduced from other continents had a lower diversity of ECM fungi than was suggested by the importance of these trees in landscapes. Ecological implications of specificity of host fungal relationships are considered in section 5.2. 3.4. Pollution and climate Numerous reports attest to altered levels of ECM inoculum and/or diversity at field sites affected by anthropogenic pollution from industrial or urban sources (e.g. Danielson and Pruden 1989; Tosh et al. 1993; Kieliszewksa-Roikicka et al. 1997). Because some of these studies were conducted without quantification of soil pollutant status, and since a complex range of pollutants is generally present at such sites, it is difficult to infer any cause and effect relating to ECM fungal diversity and pollutants from these data. A clearer picture has arisen from simple glasshouse- and field-based experiments that have considered the effects of pollutants on ECM associations either singly or in combination (Cairney and Meharg 1999). These studies provide strong evidence that most forms of pollution can result in decreased ECM infection and altered below-ground community structure, although such effects may vary with the level and duration of exposure to the pollutant(s). Nitrogen deposition can lead to a decrease in percentage total root colonisation by ECM fungi (e.g. Tétreault et al. 1978; Taylor and Alexander 1989; Haug et al. 1992; Taylor et al. 2000), although such decreases may be rather short-lived, disappearing within a few years of soil treatment (Arnebrant and Söderström 1992; Kårén and Nylund 1997; Nilsen et al. 1998). Nitrogen fertilisation can also shorten the lifespan of mycorrhizal roots (Majdi and Nylund 1996). Nitrogen addition can also profoundly affect the below-ground structure of ECM fungal communities. Taylor and Read (1996) reported a clear pattern of decreased ECM morphotype richness on Picea spp. hosts was associated with increased nitrogen deposition across Europe. Moreover, they observed a change from those ECM fungi that can readily utilise organic nitrogen in favour of those which rely largely or solely upon inorganic nitrogen sources, at sites where nitrogen deposition was greatest. Smaller-scale studies also support marked shifts in ECM fungal community structure in response to nitrogen deposition, with decreases in the relative frequency of particular ECM fungi noted following nitrogen fertilisation (Taylor and Alexander 1989; Kårén and Nylund; 1997). The form of nitrogen in soil may further differentially influence below-ground ECM fungal community structure (Arnebrant and Söderström 1992). Significantly, the data of Arnebrant and Ectomycorrhizas in plant communities 121 Söderström (1992) were collected 13 years following fertilisation and indicate that nitrogen-mediated changes to ECM fungal communities may have more long-term ecological relevance than those observed for overall ECM colonisation. Arnebrant (1994) has shown that intraspecific differences exist in the sensitivity of ECM extramatrical mycelial systems to nitrogen additions, suggesting that growth of some fungi through soil can be profoundly affected by nitrogen inputs, while others appear relatively insensitive. Differences of this nature are likely to strongly influence the relative competitiveness of ECM fungi (Arnebrant 1996) and may underpin nitrogen pollution-related shifts in structure of belowground communities. The influence of acid deposition on forest trees and their associated ECM fungi has received considerable attention. Some field and glasshouse investigations indicate that acid deposition may significantly decrease percentage ECM infection (e.g. Danielson and Visser 1989; Esher et al. 1992; Stroo et al. 1988), while others report no obvious correlation between the two (e.g. Nowotny et al. 1998; Adams and O’Neill 1991). Frequently-cited reports of changes in ECM morphotype assemblages resulting from acidification (e.g. Gronbach and Agerer 1986; Roth and Fahey 1998), however, provide convincing evidence that soil acidification effects below-ground ECM fungus communities. Notably several studies recorded a decline in ECM fungal taxa that produce extensive mycelial systems in soil (Dighton and Skeffington 1987; Markkola et al. 1995). Toxic metal pollution may similarly reduce ECM infection and alter below-ground community structure (e.g. Chappelka et al. 1991), as may a range of organic chemical pollutants (e.g. Nicolotti and Egli 1998). Interactive effects between pollutants may further influence the structure of ECM fungal communities (see Cairney and Meharg 1999). In contrast to other forms of pollution, elevated atmospheric CO2 concentrations can increase percentage ECM infection of coniferous and hardwood hosts (e.g. Norby et al. 1987; Godbold et al. 1997). This effect may be short-lived under some conditions (e.g. Runion et al. 1997; Walker and McLaughlin 1997; Walker et al. 1999b) and there may be strong interactive effects with edaphic conditions such as soil nutrient and/or moisture status and atmospheric temperature (Conroy et al. 1990; Delucia et al. 1997; Tingey et al. 1997). Recent work reveals profound effects of CO2 enrichment on ECM fungal communities, with some taxa being positively influenced at the expense of others. Specifically, these studies suggest shifts in community structure in favour of taxa that produce extensive extramatrical mycelial systems in soil (Godbold and Berntson 1997; Godbold et al. 1997; Rey and Jarvis 1997). Single ECM fungi are also known to produce more substantial mycelial systems under elevated CO2 concentrations (Ineichen et al. 1995), the implication being that some ECM fungi may be carbon-limited at ambient CO2 concentrations and that increased carbon availability favours taxa that produce more substantial mycelia. Ectomycorrhizal fungi are a major pathway for carbon flow into soils and the magnitude of this pathway can 122 Microorganisms in Plant Conservation and Biodiversity be increased by elevated CO2, at least in some cases (Runion et al. 1997; Walker et al. 1999b). The distribution and mycorrhizal efficacy of fungi forming ECM associations is influenced by climatic and edaphic factors (Slankis 1974; Smith and Read 1997). These fungi are generally considered to be acidophilic (preferring a low soil pH) inhabitants of litter layers near the soil surface (A horizon), but some “early stage fungi” (Section 4.1) prefer mineral soils which may be calcareous. Tyler (1992) found that for macrofungi in a European forest (dominated by the ECM tree Fagus), the relative importance of ECM fungi increased (and saprobes decreased) in more-acidic soils. In this study, the distribution of many fungi was correlated with edaphic factors, such as soil organic matter and metal ion content. Isolates of ECM fungi show considerable inter- and intraspecific variations in responses to the factors listed in Table 4. Isolates of ECM fungi from polluted soils often have higher tolerance to metal ions under experimental conditions, than isolates of the same species from normal soils and thus may help their hosts to survive in these conditions (Hartley et al. 1997; Meharg and Cairney 2000a). However, results obtained from in vitro experiments are often poorly correlated with responses to similar factors in soils (Cline et al. 1987; Coleman et al. 1989; Hung and Trappe 1983; Hartley et al. 1997). It has been suggested that variations in tolerance to edaphic factors may restrict geographic ranges of fungi, influence the outcome of fungal competition, or responses to factors such as drought (Parke et al. 1983a; Last et al. 1984; McAfee and Fortin 1986). It is apparent that a wide range of variation in tolerance to edaphic and climatic factors (such as temperature extremes, drought, soil toxicity etc.) often occurs, both between and within species of mycorrhizal fungi and that this variation likely represents adaptation to specific site conditions (Trappe 1977; Trappe and Molina 1986). 4. Natural ecosystems 4.1. Fungal communities Forest plant communities that host ECM fungi are often relatively species-poor, however fungal species richness within these forests is characteristically high (Malloch et al. 1980; Allen et al. 1995). Goodman and Trofymow (1998); for example, estimated that up to 100 ECM root morphotypes (considered to representing either species or genera) were present in four 0.36 ha plots of an old-growth Canadian Douglas fir stand. Similar estimates have been derived from ITS-RFLP and /or morphotype data for stands of a range of other forest types dominated by coniferous, deciduous, eucalypt, or dipterocarp trees (Pritsch et al. 1997; Gehring et al. 1998; Goodman and Trofymow 1998; Ingleby et al. 1998; Glen et al. 1999; Jonsson et al. 1999a). It must be stressed that there are also some ECM forest habitats in which fungal diversity is low. These include, Alnus rubra stands in North America which host only a handful of ECM fungal taxa (Miller et al. 1991), and Pisonia grandis on coral cays in the western Indian and eastern Pacific oceans – which may Ectomycorrhizas in plant communities 123 be associated with a single ECM taxon across its entire geographical range (Ashford and Allaway 1985; Chambers et al. 1998). The diversity of ECM fungi in recently-disturbed habitats is typically much lower than in undisturbed sites and there are some species which are characteristically found in disturbed sites (Danielson 1985; Mason et al. 1987; Jumpponen et al. 1999). Reductions in fungal diversity from disturbance likely result because many fungi are eliminated, do not have resistant propagules, or have a limited capacity to adapt to large changes to their environment (Section 2.4). Fungal succession occurs under maturing stands of trees as a few pioneering fungi are gradually replaced by increasing numbers of fungi which typically fruit in older habitats (Gardner and Malajczuk 1988; Termorshuizen 1991; Richter and Brun 1993; Keizer and Arnolds 1994; Lu et al. 1999). Visser (1995) examined species richness in regenerating Pinus banksiana stands following fire. For the first six years, roots were colonised by relatively few fungi, but diversity increased markedly in 41 year-old and 65 year-old stands, the latter having a broadly similar community to a 112 year-old stand. Lu et al. (1999) observed that the diversity of ECM fungi fruiting in Eucalyptus globulus plantations in Western Australia steadily increased by approximately two species per year (Figure 3). In another study of ECM fungal successions on previously cultivated land, ECM species richness increased until canopy closure and then declined (Dighton and Mason 1985). Fungal diversity generally increases until late in succession, when the number of species present may decline when fungi with more specialised host or substrate preferences predominate (Bills et al. 1986; Last et al. 1984). Figure 3. Relationship between the number of species of putative ECM fungi fruiting in E. globulus plantations in WA and the age of trees (data are from Lu et al. 1999) (r2 = 0.818, P < 0.01). Observations of fungal succession in aging tree stands have resulted in the designation of two groups of fungi which occupy separate ends of this continuum. Fungi which typically associate with young trees in disturbed habitats or plantations have been termed early-stage fungi, while those that associate with old trees are termed late-stage fungi (Dighton and Mason 1985). Most studies of tree monocultures in disturbed habitats have supported this concept (e.g. Chu-Chou and Grace 124 Microorganisms in Plant Conservation and Biodiversity 1982; Gardner and Malajczuk 1988; Cripps and Miller 1993; Visser 1995; Lu et al. 1999). However, there are cases where succession in young forests does not start with early stage fungi (Newton 1992; Keizer and Arnolds 1994; Helm et al. 1996; Bradbury et al. 1998). Early stage ECM fungi generally are easier to introduce into disturbed sites than late stage fungi (Danielson 1985; Lu et al. 1999). Typical examples of early stage fungi that are often observed in young plantations include members of the genera Pisolithus, Scleroderma and Laccaria. The factors underlying ECM fungal species richness in forest habitats have not been elucidated but, spatial and/or temporal resource partitioning, along with patterns of disturbance and competition may all play a role (Bruns 1995). A number of soil factors, including host plant age and physiological status, soil microbes, litter accumulation, fungal competition and inoculum availability could drive mycorrhizal fungus succession (Danielson 1985; Keizer and Arnolds 1994; Bruns 1995; Smith and Read 1997; Lu et al. 1999). Early stage ECM fungi typically grow in disturbed mineral soils with low organic matter with young host plants, while late stage fungi occur in the litter layer of mature forest soils (Mason et al. 1987; Gardner and Malajczuk 1988; de Vries et al. 1995; Lu et al. 1999). Physiological differences between early and late stage fungi are also apparent in aseptic culture experiments (e.g. Gibson and Deacon 1990). While we do not fully understand the factors influencing changes in ECM species richness with stand age, it is likely that changing soil properties and/or altered patterns of carbon allocation associated with tree maturation play a significant role. Spatial variability in soil properties and fungal population structure are also likely to be important in forests with high ECM fungal diversity. For example, of the 2000 or so fungi which associate with Douglas fir throughout its range, only about 10% of these will be found at any one location (Helm et al. 1996). Some of this high beta diversity may be due, in part, to spatial variations in soil properties which influence the competitiveness of individual fungi. However, it is likely that local variations in site histories and fungal dispersal events were responsible for initiating this spatial variability, which is maintained by processes we do not yet understand. Although generally species-rich, below-ground communities of ECM fungi are characteristically dominated by a small number of common taxa (e.g. Gehring et al. 1998; Horton and Bruns 1998; Jonsson et al. 1999b). These locally-abundant taxa, which inhabit a large proportion of available roots and presumably explore a proportionately large soil volume, may be functionally dominant (Horton and Bruns 1998), but, this remains to be demonstrated. Indeed, while ECM fungal diversity is widely regarded as important in ecosystem functioning and forest sustainability (e.g. Jones et al. 1997; Pritsch et al. 1997), the extent of functional diversity within the taxonomically diverse ECM fungal communities is currently unknown. It is likely that many ECM fungal taxa fulfil broadly similar ecological roles and, as such, that a high degree of functional redundancy exists (Allen et al. 1995). Attempts have been Ectomycorrhizas in plant communities 125 made to group taxa based on their abilities to utilise various substrates as sources of nutrients such as amino acids, which some ECM fungi can utilise but others cannot (Abuzinudah and Read 1986; Gebauer and Taylor 1999). It is known that substantial inter- and intra-specific variations occur between ECM fungi in their responses to environmental conditions (Table 4 and see Cairney 1999), growth and survival strategies (Section 2.2), etc. Our relatively poor understanding of ECM functioning in natural ecosystems, severely hampers our ability to predict the extent to which disturbance, pollution and other stresses will influence functioning of ECM-dominated plant communities. 4.2. Plant communities Mycorrhizal associations could influence plant community structure, by affecting the richness or evenness of populations of coexisting plants, or by changing the competitive ability of species. Table 7 in Chapter 4, shows 4 categories of intraspecific and interspecific interactions involving plants with different mycorrhizal requirements and association types. Facultatively mycorrhizal plants are not considered here, as plants with ECM generally are highly dependent on these associations (Section 3.2). Interactions between non-mycorrhizal plants are considered in Chapter 4. The impact of ECM associations on competitive interactions between plants would differ if competitors have (1) the same type of association, or (2) different types of associations. These cases are considered separately below. 4.2.1. Interactions between plants with ECM When growing together, plants with the same type of mycorrhizas are likely to be more equal competitors than plants with different types of mycorrhizas (Newman 1988). Plants with ECM presumably compete with other ECM plants for the same pools of soil resources (forms of nutrients), but plants with other nutrient uptake strategies (e.g. AM, ericoid mycorrhizas, non-mycorrhizal roots) may access different forms of nutrients. Competition between ECM hosts is more complex than between plants with AM (Chapter 4, Section 4.2), because ECM fungi vary more widely in their capabilities (e.g. to access organic nutrients) than AM fungi. Also, many ECM fungi are specific to certain hosts (Section 3.3), while AM fungi generally are non-specific. When different species of ECM plants grow together, the nature of competition will differ if they share the same (broad host range) fungi with a common pool of mycelia, or have separate host-specific ECM fungi that compete for soil resources. Finlay (1989) and Perry et al. (1989b) found that fungi which associate with two competing hosts, allow both to grow well together, but more specific fungi stimulated the growth of one host to the detriment of the other plant. The presence of hostspecific fungi in these experiments shifted the balance of nutrient competition in favour of their hosts. However, these experiments were conducted with only a few fungi, while plants in nature normally associate with a diverse mixture of broad and narrow host range fungi, so 126 Microorganisms in Plant Conservation and Biodiversity the impact of any one fungus on plant competition is likely to be much less dramatic in the field. Nevertheless, we can postulate that the evolution of specific host-fungus interactions may have occurred to provide benefits during nutrient competition and is more likely to occur for dominant trees in forest communities. Broad host range ECM fungi can form mycelial connections between different host taxa in the field, facilitating bi-directional transfer of carbon between them and resulting in a net carbon gain by one host (Simard et al. 1997). Common hyphal networks may assist the establishment of seedlings which share mycorrhizal partners with dominant trees. It has often been suggested that seedlings growing under mature trees of the same species are supported in this way (Newman 1988; Smith and Read 1997). This may explain why Pseudotsuga trees usually become established in patches of the ectendomycorrhizal shrub Arctostaphylos, but not under AM plants (Horton et al. 1999). A delta 13C study has shown that most of the carbon received by shared ECM fungi comes from the overstorey trees, which help to support understorey species (Högberg et al. 1999). Preservation of ‘guilds’ of interconnected host plants (Perry et al. 1989a) and their associated fungal partners may help support the sustainability of ECM plant communities. Molina and Trappe (1982) have suggested that plants such as Alnus rubra and Pseudotsuga menziesii, which often form pure stands during early succession, tend to form specific associations with ECM fungi, while species such as Tsuga heterophylla, which become established in the understorey of other trees, usually have non-specific ECM associates. Thus, the availability of particular strains specifically required by different hosts could be a regulating factor during plant succession in some habitats. Despite the evidence provided above, the ecological importance of carbon and nutrient transfer between interconnected plants in natural ecosystems is not clear. The fact that only a very small proportion of mycorrhizal seedlings survive to become trees, demonstrates that help provided by mycelial interconnections is generally not sufficient to affect the outcomes of competition (Newman 1988; Brundrett 1991). The greatest impact of sharing a common type of mycorrhiza appears to be an increase in the functional similarity of the roots systems of different species, so they are more equal competitors for soil nutrients which limit plant growth (Brundrett 1991). Even if the magnitude of below-ground carbon movements along hyphal interconnections is not sufficient to influence survival and growth of subordinate taxa, these interconnection can still function as a form of Cupertino where plants help each other by supporting a common mycelial network. The most extreme examples of resource transfer between plants with common mycorrhizal fungi are non-photosynthetic plants in the Orchidaceae and Monotropoideae (Ericaceae). Plants such as Monotropa live entirely by tapping into mycorrhizal fungus networks supported by connected autotrophic plants (Björkman 1960; Furman and Trappe 1971). Associations of non-photosynthetic plants appear to be more Ectomycorrhizas in plant communities 127 parasitic (from the fungal perspective) than mutualistic. These plants apparently have evolved a high degree of specificity in their associations with ECM fungi (Cullings et al. 1996; Taylor and Bruns 1997, 1999). 4.2.2. Interactions between different types of mycorrhizal associations Vegetation dominated by plants with one mycorrhiza type apparently can be a hostile environment for plants with other associations to grow in. It has been reported that trees with ECM often fail to become established in sites dominated by plants with ericoid mycorrhizas such as shrublands of Gaultheria, Kalmia, Rhododendron or Calluna heathlands (Robinson 1972; Messier 1993; Yamasaki et al. 1998; Walker et al. 1999b). Restricted availability of mineral nutrients and reductions in ECM fungus activity were reported within these habitats. Robinson (1972) suggested that an allelopathic inhibition of ECM fungi by exudates of Calluna contributed to nutrient deficiency problems for tree seedlings in heathlands. Examination of the world-wide distribution of plant communities reveals that most forests are dominated by trees which form ECM or AM associations and forests where both types of trees are equally dominant are rare (Brundrett 1991; Allen et al. 1995; Smith and Read 1997). It has been proposed that ECM-tree dominated plant communities are more likely to occur in soils with high organic matter and a predominance of organic nutrients, while AM-trees are more likely to dominate in mineral soils (Section 3.1). These relationships may explain the predominance of ECM forests in cooler climates. The situation in tropical regions is more complex as there do not appear to be major differences in soil properties between ECM and AM dominated forests in the same regions (Högberg 1986; Högberg and Alexander 1995; Newbery et al. 1997; Moyersoen et al. 1998). In eucalypt-dominated forest in Western Australia, roots of plants with ECM, AM or non-mycorrhizal cluster roots tended to be distributed in different soil patches, so may avoid direct competition for nutrients (Brundrett and Abbott 1995). Changes to soil properties apparently result because host trees produce leaves which are highly resistant to decomposition, resulting in slower nutrient cycling and a predominance of organic nutrient sources which are more accessible to ECM than AM fungi (Girard and Fortin 1985; Allen et al. 1995). It has also been proposed that substances in the leaf litter of ECM plants such as pine trees can inhibit AM fungi (Tobiessen and Werner 1980; Kovacic et al. 1984). Plant communities dominated by ECM plants may have a tendency to be self-perpetuating, by producing a soil environment which is hostile to AM fungi. Some plants with AM have also been reported to adversely affect ECM fungi. Hanson and Dixon (1987) found that ECM fungi could reduce the impact of allelopathy due to fern frond leachates on oak (Quercus rubra) seedlings. The abundance of weeds with AM associations can influence ECM formation by pine trees in plantations (Sylvia and Jarstfer 1997). Colonisation by AM fungi reduces the lifespan of roots of Populus, a tree which predominantly has ECM 128 Microorganisms in Plant Conservation and Biodiversity associations (Hooker et al. 1995). Allelopathic interactions between competing plants are considered to be common in plant communities, but the role of mycorrhizal fungi in these interactions has rarely been investigated. A second form of competition between different types of mycorrhizal fungi occurs within the root systems of plants which are hosts to two types of mycorrhizal associations. In Australia, plants with ECM associations usually also have some VAM in their roots. These plants include major species used in plantation forestry belonging to the genera Casuarina, Allocasuarina (Casuarinaceae), Eucalyptus, Melaleuca (Myrtaceae) and Acacia (Mimosaceae) (Brundrett 1999). Plants with dual ECM/VAM associations are less often reported from other parts of the world (Brundrett 1991), but there are exceptions such as Alnus, Populus, Salix and Uapaca (Lodge and Wentworth 1990; Arveby and Granhall 1998; Moyersoen and Fitter 1998). Most gymnosperms with ECM are highly resistant to AM fungi, but their roots may contain vesicles and hyphae if grown with a companion AM plant (Hooker et al. 1995; Smith et al. 1998). A succession from VAM to ECM associations in Eucalyptus and Alnus roots often occurs as trees age in field soils (Gardner and Malajczuk 1988; Bellei et al. 1992; Oliveira et al. 1997; Arveby and Granhall 1998). Ectomycorrhizal fungi have also been observed to gradually displace VAM in Eucalyptus roots in the glasshouse (Lapeyrie and Chilvers 1985; Chen et al. 2000). Plants which can form both ECM and AM associations occur in some ecosystems. For these plants, it seems that AM are most important for their young seedlings, perhaps due to the time required for ECM fungus dispersal and establishment, while ECM usually becomes dominant when they grow older. There are plants, such as some Acacia species, which are highly receptive to ECM and AM associations throughout their lives, but this is rare and most species show a clear preference for a single type of mycorrhiza (Brundrett 1999). 4.3. Animals The roles of animals as consumers and dispersal agents of ECM fungi are considered in section 2.3. Tree-mycorrhizal fungus-dispersing animal interrelationships are important in forests, especially in western North America and Australia (Maser and Maser 1988; Claridge et al. 1992). Animals which disperse ECM fungi facilitate ecosystem recovery after disturbance (e.g. Claridge et al. 1992; Cázares and Trappe 1994; Johnson 1995; Reddell et al. 1997; Cázares et al. 1999). Ectomycorrhizal fungi and their host plants must also be considered during any attempts to manage mycophagous animal species. For example, the occurrence of hypogeous ECM fungi in different vegetation types is thought to limit the distribution of the Northern Bettong (Bettongia tropica) which needs these fungi for food in the dry season (Johnson 1996). The importance of animals as vectors for ECM fungi is suggested by the evolution of truffle-like fruit bodies in most families of ECM fungi in Australian eucalypt forests (Bougher and Lebel 2001). These Ectomycorrhizas in plant communities 129 subterranean fruit bodies would only have a selective advantage in habitats where animal dispersal of fungi is more effective than airborne spore dispersal. Marsupials which consume spores are efficient dispersal agents, because they forage near trees over a wide area and have commensal dung beetles which bury their spore-laden faeces (Johnson 1996). Unfortunately, many Australian mycophagous marsupials are now extinct throughout most of their former ranges, and this could effect ecosystem functions, especially the capacity for recovery after disturbance. We must consider secondary symbiotic associations, such as the animal vectors of ECM fungi, as well as the primary tree-fungus associations when we monitor the quality of remnant vegetation, or attempt to restore plant communities. 5. Utilising mycorrhizas This section will focus on plant conservation and ecosystem restoration. However, there also are concerns about the conservation of ECM fungi in regions where their populations are declining due to changes to soil conditions caused by pollution or over-collection by humans (Arnolds 1991; Boujon 1997; Hosford et al. 1997). Ectomycorrhizal fungi exhibit functional diversity and are adapted to local environmental and soil conditions (Table 4). Consequently, it would be important to conserve representatives of all habitats and soils within regions to maintain the functional diversity of mycorrhizal fungi and other organisms. Conservation of particular ECM fungi may also be required for nonphotosynthetic plants in the Ericaceae (Monotropoideae) which are wholly dependent on a type of ECM fungi and have specific fungal partners (Cullings et al. 1996; Kretzer et al. 2000). 5.1. Ecosystem restoration Mycorrhizal technology can be designated as any artificial means of introducing fungi to new habitats or manipulating existing populations of fungi. Most work on the introduction of ECM fungi has been for plantation forestry, or for valuable edible fungi such as the black truffle (Tuber melanosporum). Inoculation technologies have been developed to introduce fungi in the nursery or field, using soil, spores or mycelia produced in sterile culture as inoculum (Brundrett et al. 1996c). Before promoting mycorrhizal technology, it is necessary to evaluate its relative costs and benefits (Brundrett 2000). Costs arise from the resources and time required to acquire appropriate fungi, produce and apply inoculum and implement quality control measures to confirm inoculated fungi are present. Benefits primarily concern short-term increases in survival and/or growth of plants. Some ECM fungi may offer added benefits by influencing the activities of other soil microorganisms, or degradation of persistent organic pollutants in soil (Meharg and Cairney 2000b). Longterm benefits from increased mycorrhizal fungus biomass or functionality are suggested in the literature, but have not been measured in the field (Section 3.1). Potential environmental costs that may arise from introductions of mycorrhizal fungi into different locations should 130 Microorganisms in Plant Conservation and Biodiversity also be considered (Section 5.2). When ECM fungi are introduced to a site (usually by planting seedlings inoculated in the nursery), their success depends on their ability to spread through the soil to new roots and the outcome of competition with indigenous fungi (Last et al. 1984; McAfee and Fortin 1986). Hostfungus specificity is also important, as poorly compatible fungal isolates will often fail to establish. Cases where substantial improvements in tree growth due to mycorrhizal inoculation were measured are outnumbered by trials where there was no measurable response (Castellano 1994; Jackson et al. 1995; Brundrett 2000). Nevertheless, there have been cases where ECM fungus inoculation has resulted in significant growth enhancement in the field, especially where trees have been grown in disturbed habitats such as mine sites, or exotic locations with few compatible fungi (Malajczuk et al. 1994; Castellano 1994; Reddell et al. 1999). Responses to mycorrhizal inoculation are also highly dependent on soil conditions, especially soil fertility. For example, Cistus incanus fails to establish in infertile calcareous soils without mycorrhizal fungi, but grows well without fungi if fertilised (Berliner et al. 1986). Mycorrhizal fungi may be an important consideration in rare species recovery programs. They sometimes are included in cultivation attempts as an insurance policy to eliminate nutritional factors as a cause of failure. Inoculation of tissue cultured plants is especially important for subsequent plant survival and growth during critical early stages of establishment in soil (Martins et al. 1996; Reddy and Satyanarayana 1998). 5.2. Potential problems with fungal technology While ECM inoculation programs were required for the successful establishment of plantations of trees such as pines and eucalypts at exotic locations, they have the effect of introducing alien ECM fungal taxa along with their hosts. It is possible that alien fungi may influence local ECM fungal diversity via invasion of native forest systems. The extent to which this occurs will depend upon the ability of the introduced fungi to persist at the exotic sites and the extent to which they are able to form ECM with the native vegetation. There is certainly evidence that some introduced ECM fungi can persist in plantations at exotic locations for many years following introduction (e.g. Martin et al. 1998; Selosse et al. 1999). It has generally been assumed that no environmental costs will arise from introductions of mycorrhizal fungi into different geographic locations. The host specificity of many fungi often prevents fungi from associating with indigenous hosts belonging to other families (Section 3.3). However, introducing fungi that are more, or less effective symbiotic partners than indigenous strains, may impact on plant community structure, by influencing the outcome of competition between plants. We would expect the potential for broad host range fungi to invade indigenous vegetation should be greater than for fungi which associate with few host plants. Fungi associated with Eucalyptus spp. are a useful Ectomycorrhizas in plant communities 131 example in this context. The geographical isolation of the Australian flora is considered to have enabled co-evolution of highly specific ECM associations within the genus Eucalyptus, that are poorly compatible with tree species from other continents (Malajczuk et al. 1982, 1990; Bougher 1995). Recent investigations in Kenya, for example, indicate that Pisolithus species introduced during the past 100 years into Eucalyptus and Pinus plantations are only found in association with their respective hosts (Martin et al. 1998). Australian species of Laccaria, Hydnangium and Hysterangium associated with eucalypt plantations at exotic sites also appear to remain confined to that genus (Castellano 1999). None of the ECM fungi introduced to Australia with Pinus are known to have spread into Eucalyptus forests (Molina et al. 1992; Bougher 1994; Dunstan et al. 1998). However, the pine fungus Amanita muscaria is now found under Nothofagus in Tasmania and New Zealand (T. May pers. com., P. Johnson and P. Buchanan pers. com.). Fungi which readily switch to new families of host plants include several Amanita species which have crossed over to eucalypts introduced to North America and Europe (Brundrett and Bougher 1999). These relatively promiscuous fungi apparently can invade indigenous forests as “weeds” that compete with indigenous species. Perhaps we should be concerned about some feral fungi, such as extremely toxic Amanita species, that have the potential to invade native forests and kill indigenous fungus-feeding animals. 6. Conclusions This review attempts to summarise key roles of ECM fungi in natural ecosystems and provide information that should be of value to people who study processes in or help to conserve natural habitats. Plants with mycorrhizas are dominant in most communities (Table 1 in Brundrett and Abbott, this volume). Thus, mycorrhizal fungi typically are the primary soil interface for plants and must be considered in all studies of nutrient cycling or impacts of nutrient supply on plant productivity or diversity. Processes mediated by ECM associations in natural ecosystems are listed in Table 6. There is much scope for future ecological work investigating the processes listed in Table 6. In these studies, the first challenge is to determine what type of mycorrhizal associations the plant(s) being considered have. Information about mycorrhizal associations has been summarised for some locations such as the UK (Harley and Harley 1987) and Australia (Brundrett 1999). Information exists for many other locations, but may be harder to find. Published lists must be expected to contain some errors or misinterpretations and contradictory data exists for some species. Consequently, it will often be necessary for researchers to examine roots of their plant species using microscopic techniques to confirm the presence of mycorrhizal associations (Brundrett et al. 1996c). The second challenge is to determine if mycorrhizal fungi are already present in soils where host plants will be grown. Sampling methodologies exists for detecting inoculum of these fungi (Brundrett et 132 Microorganisms in Plant Conservation and Biodiversity al. 1996c). However, we would expect ECM fungi already to be present in most habitats, provided compatible hosts occur nearby and soils have not been substantially altered by disturbance, pollution, etc. A third challenge may be to determine if it is appropriate to introduce fungi to sites and when it is unnecessary or even harmful to do so (Section 5.2). Ectomycorrhizal fungi can be dispersed by wind and animals and may rapidly arrive in new sites. Some host plants may also grow well initially without ECM fungi provided AM fungi are present or soils are relatively fertile. Table 6 . Ecological implications communities. of mycorrhizal associations in plant 1. The absence of mycorrhizal fungi is likely to impact on plant survival, growth, reproduction, stress tolerance, disease resistance and water relations. 2. Environmental factors or disturbance events which impact on the abundance and spatial distribution of mycorrhizal fungus inoculum are likely to affect the diversity and structure of plant communities. 3. Different forms of nutrient competition occur between plants with different mycorrhiza types and also between plants with different associated fungi. 4. Plant communities dominated by plants with different mycorrhiza types are likely t o have different nutrient cycling processes in their soils. 5. Environmental factors and/or soil conditions determine where plants with different mycorrhizal strategies are dominant. 6. Plants with different mycorrhizal strategies, or associated with different fungi will vary in their capacities to tolerate adverse soil conditions listed in Table 4, which may result from pollution, climate change, etc. 7. Mycorrhizas may ameliorate plant competition by increasing the functional similarity of roots and by interconnecting plants of the same or different species. 8. Succession from ruderal species to a climax plant community often involves changes to the dominant mycorrhizal types of plants and/or populations of mycorrhizal fungi. 9. Communities dominated by plants with a particular mycorrhiza type can be a more hostile environment to plants with other types of association than we would expect if we only consider aboveground competition. 10. Secondary associations between ECM fungi and animal dispersal agents may influence the establishment of host plants, especially in disturbed habitats. 11. Any factors which impact on the taxonomic and functional diversity of ECM fungi may eventually affect the productivity and structure of plant communities. We hope that we have provided some guidance about where ECM associations need to be considered in scientific studies of ecosystems or individual species. This information would also be relevant to the management of plants, plant communities and mycophagous animals. These fungi have many important roles (Table 5) and the fruit bodies of some species are important as food sources for animals and humans. The importance of ECM associations was first proposed by Frank in 1885. Since then, we have learned how to manipulate these associations in experiments and have amassed a substantial body of information about Ectomycorrhizas in plant communities 133 their roles in plant nutrition. However, there is still much to learn about ECM in natural ecosystems, especially concerning: (i) How they acquire soil nutrients from organic and inorganic sources in co-operation/competition with other organisms in soil food webs. (ii) The magnitude of nutrient and carbon transfer between connected individuals of the same and different species and the role of these transfers in ecosystems. (iii) The impact of ECM associations on the disease resistance, water relations, etc. of their hosts. (iv) The influence of changes to soil structure and chemistry caused by ECM fungus hyphae (e.g. hyphal mats, carbon storage, weathering of minerals, etc.) on plant nutrient supplies. (v) The role of taxonomic and functional diversity in these fungi. (i.e. Why there are many species of ECM fungi in some habitats and few in others?) Perhaps the start of a new millennium is an appropriate time to reconsider where we heading. It is time to stop repeating the same basic experiments demonstrating benefits provided to yet another plant species using highly simplified experimental conditions. Instead we need to shift focus to the roles of ECM in-situ in natural ecosystems, by considering the neglected areas of research listed above. 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