Ectomycorrhizae and tree seedling nitrogen nutrition in forest restoration
Author
B. J. Hawkins,
Melanie D. Jones,
J. M. Kranabetter
Abstract In natural environments, tree roots are almost always in intimate, symbiotic association with particular species of fungi through the formation of mycorrhizae. Most mycorrhizal fungi provide soil resources, particularly nitrogen (N), phosphorus and/or water to the tree, and can increase the abiotic and biotic stress resistance of their hosts. The fungi benefit by receiving fixed carbon from the tree. The association is of particular benefit on harsh or degraded sites. This review surveys recent literature on ectomycorrhizal (ECM) associations of temperate and boreal forest trees as it relates to N-nutrition and restoration of forests on sites where native mycorrhizal communities have been altered or depleted. Part I emphasizes the ECM fungal partners. Changes in ECM communities through primary and secondary succession are reviewed and related to the influence of N availability. The effect of N-related functional traits of ECM fungi on their distribution is discussed. Part II focuses on the ECM plant partners. The influence of ECM fungi on plant N uptake, and effects of N deposition and fertilization are presented. The benefit of ECM inoculation under different disturbance regimes and the benefit of greater ECM diversity are reviewed. Variations among and within tree and ECM fungal species in the forms of N taken up and utilized are highlighted. Conclusions include recommendations for including ECM fungi in forest restoration projects. Part 1 Introduction
Low nitrogen (N) availability in marginal soils or recalcitrant litter is one of the major factors limiting plant growth in temperate and boreal forest ecosystems (Read et al. 2004; Rennenberg et al. 2009). After severe disturbance, successful reestablishment of tree cover on N-limited sites must consider tree nutrition and practices that optimize N acquisition by trees. While investigations of tree nutrition often focus on above-ground indices of growth and foliar nutrient concentrations, these parameters are directly linked to the below-ground world of roots, soil and soil microbes. Roots of trees in their native environments are always in intimate association with particular species of fungi through the formation of mycorrhizae (Smith and Read 2008). Mycorrhizal fungi play a critically important role in uptake of N, phosphorus (P) and other nutrients, and in nutrient transfer to the plant (Chalot and Plassard 2011), yet they are difficult to study because they are challenging to sample in their hyphal phase, to identify at the species level, to culture and to manipulate. Increasing access to molecular tools over the past 20 years has facilitated the study of mycorrhizae and given us insight into the diversity of fungi in forest ecosystems (Horton and Bruns 2001; Courty et al. 2010). This paper aims to provide an overview of recent literature on ectomycorrhizal association of temperate and boreal forest trees as it relates to N-nutrition and forest restoration. While many studies focus on mycorrhizal associations of forest trees in reforestation situations, there is much less information on the dynamics and benefits of mycorrhizal association in forest restoration. In this review, we attempt to maintain a focus on forest restoration on harsh or degraded sites where the native mycorrhizal community has been depleted or lost, and to use information from forest regeneration studies to infer ecosystem responses under conditions of more severe disturbance.
Forest restoration can be defined merely as the reestablishment of tree cover after disturbance, or more broadly as the return of a forest ecosystem to its “historical” state. A more nuanced definition includes the reinstatement of ecological processes, which accelerate recovery of forest structure, ecological functioning and biodiversity levels towards those typical of the climax forest (Elliott et al. 2013). The presence of fully functioning soil microbial communities, including fungi and bacteria, is an indicator of restoration success; however, it is more difficult to assess the role the microbial community plays in establishing plant communities, and the potential for manipulating the soil microbial community to accelerate the return to a climax state (Harris 2009). There is substantial evidence that as ecosystems mature from disturbed to late successional states and complex organic material enters the soil matrix, the ratio of fungal to bacterial biomass increases (Harris 2009). This suggests that the microbial community follows, rather than leads, the developments in the above-ground community (Harris 2009). In contrast, the kind of mycorrhizae, arbuscular, ecto- or ericoid, influences the plant community that can occupy a site, so the mycorrhizal community could be viewed as a “keystone” feature of a functioning community (Miller and Jastrow 1992). In either scenario, soil fungal communities, including mycorrhizal fungi, are a critical component of healthy forest ecosystems.
Mycorrhizae are said to be the most important symbioses on earth (Bücking et al. 2012). The word mycorrhiza is derived from the Greek mykós, “fungus” and riza, “roots”. In most mycorrhizal symbioses, fungi supply nutrients, particularly N and P, and/or water to the plant in exchange for carbon (C) in the form of sugars. At least 80 % of surveyed land plant species are mycorrhizal (Wang and Qiu 2006). Smith and Read (2008) classified mycorrhizal associations into seven types, a system that is widely accepted. Of these seven types, forest trees most commonly form ECM, arbuscular mycorrhizae (AM), or both. Approximately 65 % of plant species form AM (Wang and Qiu 2006) which may explain the focus of restoration literature on this mycorrhizal type. Although ECM associations are more rare than AM among land plant species, ECM are the most important mycorrhizal associations for temperate and some tropical trees. All species of conifers surveyed form ECM and/or AM, and almost all are obligately mycorrhizal (Wang and Qiu 2006). ECM are the main type of association for the Araucariaceae, Pinaceae, Betulaceae, Casuarinaceae, Fagaceae and Salicaeae, and are also common for the Caesalpiniaceae, Cupressaceae, Dipterocarpaceae, Juglandaceae, and Myrtaceae (Wang and Qiu 2006; Smith and Read 2008). Because of their importance to many tree species across the globe, ECM are the focus of this review.
Ectomycorrhizae are mainly formed by fungi in the phylum Basidiomycota but also by some species in Ascomycota, and are relatively closely related to saprotrophic fungi (Bücking et al. 2012; Floudas et al. 2012). Ectomycorrrhizal fungi appear to have evolved relatively recently from a number of independent origins (Hibbett et al. 2000; Wang and Qiu 2006). Several phylogenetic studies date the evolution of ECM fungi to 120–190 million years ago (Veneault-Fourrey and Martin 2013) and fossil evidence from British Columbia indicates that ECM associations were well established 50 million years ago (LePage et al. 1997). Only ~3 % of plant species form ECM associations, but in contrast to AM fungi, there are at least 7750 documented ECM fungal species and some estimates of ECM species richness range as high as 25,000 species (Rinaldi et al. 2008). These many ECM fungal species generally have a higher degree of host specificity than AM fungal species (Wang and Qiu 2006), and in terms of global species richness, host plant family has the strongest effect on the phylogenetic community composition of ECM fungi (Tedersoo et al. 2012).
In ECM associations, the fungus changes root morphology significantly. The intraradicle mycelium forms a conspicuous intercellular network, the Hartig net, surrounding individual epidermal cells in angiosperms or epidermal and cortex cells in gymnosperms, that plays a key role in nutrient transfer between fungus and plant (Bücking et al. 2012; Hacquard et al. 2013). Outside the mantle, the extraradical mycelium forms an extensive network in the soil and can represent 75 % of the potential absorbing system area and over 99 % of the nutrient-absorbing length in pine roots (Rousseau et al. 1994). Ectomycorrhizae are particularly important in N uptake and also make a significant contribution to P nutrition (Jones et al. 1998; Bücking et al. 2012; Becquer et al. 2014).
Effect of disturbance on ECM community diversity
Primary succession, a process occurring over hundreds to thousands of years, begins when a substrate devoid of vegetation and developed soil is exposed. This condition could be considered analogous to restoration of extreme sites with very little organic matter, such as mine spoils, landslides or very severe burns. Secondary succession, typically occurring over decades to centuries, occurs on a substrate that previously supported vegetation prior to a disturbance removing the plant cover. Forest harvesting, moderate fire or flooding could initiate secondary succession. When restoring extremely or moderately degraded forest sites, the suite of ECM fungi that is present will change over time with stand composition, stand age, individual tree age, or even individual root age (Dickie and Reich 2005).
Primary succession
Studies of changes in mycorrhizal diversity with primary succession often compare mycorrhizal species across a chronosequence. In the long-term chronosequence approach, changes in patterns of N and P availability over time are associated with changes in primary productivity, nutrient cycling, and plant and fungal functional traits (Dickie et al. 2013). Typically, in new substrates, N is nearly absent while P is relatively plentiful. Over time as N fixation, plant cover and soil organic matter increase, N pools increase while P availability declines (Lambers et al. 2008). Over the scale of thousands of years, nutrient limitation of primary productivity is expected to shift from N-limitation early in primary succession to co-limitation by N and P in mature phases, to P limitation in the absence of ecosystem-rejuvenating disturbance (retrogression) (Dickie et al. 2013). Although ECM plants can be expected at many stages of primary succession, Lambers et al. (2008) predicted that they are most abundant when N accumulates in organic form and where soil P has decreased. In a review of literature from long-term chronosequences across the globe, Dickie et al. (2013) found no evidence of predictable shifts in mycorrhizal type (AM vs. ECM) associated with the shifts in N and P limitation; however, typically, ECM are the first mycorrhizal fungi present in primary succession as root colonization is strongly limited by inoculum availability (Trowbridge and Jumpponen 2004). Evidence indicates that the assembly of early fungal communities is strongly influenced by stochastic events and airborne spore deposition (Jumpponen 2003; Jumpponen et al. 2012), and that the diversity of fungal species gradually increases with plant community development (Jumpponen et al. 2012). In studies of retreating glaciers in the western USA, the initial ECM community comprised fungi that tolerate low organic matter and low N availability (Trowbridge and Jumpponen 2004). The most abundant genera were Cortinarius, Inocybe and Laccaria (Jumpponen et al. 2012), genera also common in early primary succession on montane sites in Japan (Nara et al. 2003). In addition, Amphinema, Cenococcum, Tomentella and Thelephora are frequently observed in the earliest successional ecosystems but all are also found in the organic soils of mature forests, and can thus persist through the mature stages of ecosystem development (Dickie et al. 2013). These very early fungi in primary succession, which would likely be most suitable for the restoration of highly degraded sites, tend to have traits such as abundant sporocarp and/or spore production, palatable sporocarps with spores tolerant of digestion, persistent dormant spores, high spore infectivity, or rapidly growing, long hyphae (Dickie et al. 2013). Fungal species from these genera should be investigated as possible species to include in inocula when establishing trees on mine tailings and severely degraded soils. The further development of ECM fungal communities does not appear to converge over time, as is observed in plant communities (Brown and Jumpponen 2014), but is associated with plant cover and soil development, accumulation of organic matter and changing availability of N forms. Early-mid primary successional species such as Rhizopogon and Suillus often have abundant, large sporocarps and higher host-specificity than the early successional ECM group (Dickie et al. 2013), thus colonization is restricted until the appropriate plant partners are present. Boletus, Amanita, Russula and Lactarius are found at the highest frequency in mature ecosystems with substantial soil organic layers, and all have large, conspicuous sporocarps; however, some species of Russula and Lactarius can be found very early in primary succession (Dickie et al. 2013).
Secondary succession
Forest disturbance can affect diversity of plant, animal and microbial communities when it returns forests to early seral stages, and forest regeneration attempts to hasten the return to later successional stages. As ecosystems mature, communities of ECM fungi become more diverse (Dickie et al. 2013) and exploit a greater variety of N forms (Kranabetter 2014). Jones et al. (2003) found no compelling evidence that clearcutting reduces overall ECM fungal species diversity or inoculum potential for regenerating seedlings; however, more severe disturbance, such as clearcutting followed by burning or heavy site preparation, removes living vegetation, roots and litter and humus layers which can reduce ECM inoculum potential. Maintenance of the forest floor is particularly critical to maintaining fungal abundance and diversity (reviewed in Wiensczyk et al. 2002) and severe forest floor disturbance has been linked to reduced seedling survival, foliar nutrient concentrations and growth (Simard 2009). Invasion of previously forested areas by plants associated with a different mycorrhizal type, such as AM grasses (Simard 2009) or ericoid mycorrhizal shrubs (Jones et al. 2003), can also hinder restoration of ECM-associated trees. Differences among ECM fungal species in dispersal ability, lifespan, tolerance of abiotic conditions, degree of host specificity, root colonization strategy, ability to access nutrients of different forms and complexity, enzymatic capability for decomposition and hyphal foraging strategy will influence ECM persistence or recolonization of a site (reviewed in Jones et al. 2003). In regenerating Douglas-fir (Pseudotsuga menziesii Mirb. Franco) and paper birch (Betula papyrifera Marsh.) mixed forests, ECM fungal community diversity increased rapidly for 26 years following clearcutting and wildfire, after which fungal diversity stabilized at similar levels in the two disturbance types (Twieg et al. 2007; Simard 2009). ECM fungal community composition in this system did not stabilize until 65 years (Twieg et al. 2007), but was then found to overlap with the community composition of nearby 120-year-old fire origin forests (Simard 2009). In jack pine (Pinus banksiana Lamb.) stands, ECM community structure and diversity stabilized 41 years after wildfire (Visser 1995).
When restoring degraded sites, restoration of ECM fungal abundance and diversity is linked to the presence of soil organic matter. A greater degree of degradation and loss of organic matter results in a greater reduction in ECM fungal richness. As Harris (2009) concluded that the microbial community follows, rather than leads developments in the above-ground community, restoration of soil organic matter is key to the restoration of functional forest ecosystems.
Effect of N availability on the ECM community
Soil organic matter exerts its influence on ECM fungal diversity, in part, through its release of N. N availability has strong effects on composition, function and diversity of ECM fungal communities (Lilleskov et al. 2011; Kranabetter et al. 2009a), but results of studies of N effects on ECM associations contrast markedly depending on whether N is at natural, low levels or at elevated levels due to fertilization or atmospheric deposition. In many container and field fertilization or N deposition studies, increasing N availability is correlated with lower ECM species diversity and growth (reviewed in Bahr et al. 2013). Forests across Europe are subject to chronic N deposition from anthropogenic sources, which has significant implications for ECM fungal associations of restored forests in these regions. In European Scots pine (Pinus sylvestris L.) forests, fungal richness was negatively correlated with soil nitrate and plant tissue N concentrations, and the negative correlation of ECM richness with root N concentration was strongest of the 18 soil, plant and climatic factors tested (Cox et al. 2010). Similar results have been found in Norway spruce (Picea abies (L.) H.Karst.) forests where an N deposition increase from 27 to 43 kg N ha−1 year−1 decreased ECM root tip abundance, mycelial growth and species richness (Kjøller et al. 2012), and deposition rates from 2 to 20 kg N ha−1 year−1 decreased ECM extramatrical mycelium (Bahr et al. 2013). A critical deposition load estimate for ECM fungi of 5–10 kg N ha−1 year−1 has been supported (Jarvis et al. 2013).
The negative effect of N deposition on ECM fungal species diversity or abundance has been attributed, particularly, to increased levels of soil inorganic N and a suppression of fungal species able to use protein-N (Taylor et al. 2000). Stand age may interact with the effect of high N as the expected negative relationship between ECM mycelium growth and ammonium availability was found in Norway spruce forests 0–36 years of age, but not in older forests (Wallander et al. 2010). N form may also interact with the effect of high N deposition as glutamic acid applied for 8 weeks at 35 kg ha−1 increased, rather than decreased, ECM root tip abundance in black oak (Quercus velutina Lamb.) and pitch pine (Pinus rigida Mill.) relative to fertilization with inorganic N or the unfertilized control (Avolio et al. 2009). The question for restoration, of whether the benefit to plants of immediate N supply from fertilization outweighs the cost of loss of mycorrhizal diversity or abundance, is addressed in Part II.
In contrast to situations of high levels of inorganic N deposition or fertilization, an analysis of data sets from across the globe showed ECM root colonization intensity to increase in native soils with a high proportion of N (C:N ratio ~12), particularly at neutral pH (Soudzilovskaia et al. 2015). Assessments of ECM fungal community diversity on unmodified boreal forest soils with a natural range of low N availabilities also show increasing ECM species richness with increasing foliar N. In boreal subalpine fir [Abies lasiocarpa (Hooker) Nuttall] forests, ECM diversity assessed by molecular methods (Kranabetter et al. 2009a) or sporocarp surveys (Kranabetter et al. 2009b) increased with foliar N concentration, but the two studies differed slightly as species richness based on sporocarps decreased somewhat at the highest N levels (~45 kg available N ha−1) (Kranabetter et al. 2009b). In these studies, foliar N concentrations on the richest sites corresponded to those on the European low N deposition sites, and peak ECM diversity in natural boreal forests is suggested to coincide with the heterogenous supply of inorganic and organic N forms found on rich sites (Kranabetter et al. 2009a). In jack pine forests without N deposition, ECM fungal community composition from stand initiation to canopy closure also was positively correlated with soluble organic N and free amino acid-N, but not inorganic N (LeDuc et al. 2013). In contrast, in declining eucalypt forests in Australia, total ECM fungal richness was inversely related to available soil nitrate but also to available soil P (Horton et al. 2013). As both available soil N and P varied over wide ranges in that study (Horton et al. 2013), it is difficult to separate the effects of the two elements; however, a common element of all studies is that individual fungal species or genera have distinct responses to N (Kranabetter et al. 2009b; Cox et al. 2010; Horton et al. 2013). These specific responses will need to be taken into account if replanted trees are inoculated in the nursery with ECM fungi.
ECM fungal species differ in N form preference and uptake
N-related functional trait differences among ECM fungal species may determine species distribution in space and time (Koide et al. 2011; Lilleskov et al. 2011). The presence or frequency of many ECM fungal species is related to N and P gradients associated with successional stages (Dickie et al. 2013), or differences in available N or P among sites (Toljander et al. 2006; Kranabetter et al. 2009b; Horton et al. 2013). Field studies provide indirect evidence of adaptation of ECM fungal species to N form or availability (Koide et al. 2014), however, N gradients in the field are often confounded by moisture gradients or differences in plant communities, which can also determine the ECM fungal community. Field studies can be complemented by controlled environment studies of ECM response to N form and availability.
Cool temperate and boreal forest soils typically have low levels of available N, and often 50 % or more of the available N is organic N (Jones and Kielland 2002). Most organic N in forest soil solution is comprised of high molecular weight compounds, with amino acids constituting 10–20 % of the dissolved organic N pool (Jones and Kielland 2002). In some boreal forest soils, however, small molecular weight amino acids may constitute the majority (up to 80 %) of the organic N pool (Inselsbacher and Näsholm 2012). ECM fungi are able to take up both inorganic and organic forms of N. Ammonium is generally recognized as the most readily utilized source of inorganic N by most ECM fungi (Chalot and Plassard 2011) in mycelial cultures (Rangel-Castro et al. 2002; Guidot et al. 2005), in ECM roots in the lab (Fig. 1) and in the field (Clemmensen et al. 2008). In pure culture, Nygren et al. (2008) demonstrated wide variation among 68 species of ECM fungi in their mycelial growth with nitrate as the sole N source. While results of culture studies must be used with caution because they may not reflect N preferences of ECM fungi when growing in symbiosis with trees (Turnbull et al. 1995), Hobbie et al. (2008) also demonstrated higher nitrate uptake and greater inferred transfer of N from fungus to plant by Laccaria laccata (Scop.) Cooke associated with Scots pine, compared to Suillus bovinus (L.) Roussel, particularly at high nitrate supply.
Fig. 1
Mean (±SE) net flux (nmol m−2 s−1) of ammonium, nitrate and protons in non-mycorrhizal (NM) and mycorrhizal root tips of Douglas-fir seedlings collected from the field. Mycorrhizal root tips were associated with one of four ECM morphotyes (a–d) (n = 10–20). Net ion flux was measured with ion-selective microelectrodes using a MIFE™ system. Negative flux denotes efflux
In some studies, ECM seedlings demonstrate a strong preference for amino acids (glutamic acid) over ammonium (Read et al. 2004). The ability of ECM fungi to: (a) utilize proteins (bovine serum albumin), and (b) contribute significant quantities of protein-derived N to the plant partner was first demonstrated by Abuzinadah and Read (1986a, b, respectively). Abuzinadah et al. (1986) showed that growth of lodgepole pine (Pinus contorta Dougl. ex Loud) seedlings was similar on protein-N compared to ammonium-N with each of four ECM fungi; however, there was a dramatic range among ECM fungal species in their ability to grow on protein in culture (Abuzinadah and Read 1986a; Finlay et al. 1992) and provide protein-N to their plant partner (Abuzinadah and Read 1989). Utilization of N in large biopolymers could include intact uptake, or breakdown of the molecules into smaller organic or inorganic forms. ECM fungal species differ in their excreted enzyme profiles, and thus their ability for N mobilization from chitin, proteins and the myriad of organic substances found in litter and humus (Courty et al. 2005). In comparison to saprotrophs that target carbohydrate- and organic P-rich sources, the oxidative and hydrolytic enzymes of the extramatrical hyphae of ECM fungi provide greater access to recalcitrant N sources in soil (Talbot et al. 2013).
In pure culture and in association with some plants, most ECM fungi investigated are able to grow with amino acids as a N source (reviewed in Chalot and Brun 1998; Talbot and Treseder 2010) and some grow on a wide variety of amino acids (Guidot et al. 2005). Mycorrhizal roots of maritime pine (Pinus pinaster Aiton) and Norway spruce seedlings were shown to take up amino acid-N (Plassard et al. 2000; Boukcim and Plassard 2003), and amino acid uptake by high-affinity transport systems in excised, mycorrhizal European beech (Fagus sylvatica L.) roots can occur at similar or higher rates than an ammonium analogue (Wallenda and Read 1999). From double labeling studies, it appears that amino acid-derived N can be passed on to the seedling shoot, but amino-acid-derived C remains in fungal mycelium and roots (Taylor et al. 2004); however, quantum dot studies show that amino acids taken up by AM are translocated intact to the plant shoot (Whiteside et al. 2009) and the same appears to be true for ECM plants (M.D. Whiteside pers. comm.).
Differences among ECM fungal species in their ability to access and take up different N forms indicate that N form and availability may be a defining element of ECM fungal species niche (Kranabetter 2014). Evidence exists that ECM taxa are adapted to the N form predominant in their environment. In a study with seven ECM fungal species from Alaska, Lilleskov et al. (2002) showed that taxa common in soils with little inorganic N grew on protein, glutamine and serine when in pure culture, whereas species from high-inorganic N soils grew on glutamine, but poorly on protein and serine. They suggested that variation among species in proteolytic ability or responsiveness of proteolytic enzyme production may be responsible for interspecific variation in protein use. ECM fungal diversity and proteolytic capabilities are suggested to be greater in boreal podzols with mor-type humus, where N mineralization is poor and the organic N pool more diverse, than in temperate forest soils with mull-type humus (reviewed in Courty et al. 2010). Several authors have concluded that proteolytic genera well adapted to organic N uptake, such as Cortinarius, Piloderma, Tricholoma and Suillus generally decline in abundance as inorganic N availability increases, and are replaced to some degree by nitrophilic ECM genera, such as Russula, Inocybe, and Tomentella (Lilleskov et al. 2011; Jones et al. 2012; Horton et al. 2013; Kranabetter 2014). Interestingly, however, early successional ECM fungi appear to have considerable phenotypic plasticity in their ability to break down complex forms of organic N such as chitin and protein (B.A. Nicholson, unpub.).
In addition to variation among ECM fungal species in their ability to break down organic matter, differences in mycelial morphology may also contribute to differences in N form uptake (Trudell and Edmonds 2004; Lilleskov et al. 2011). Rhizomorph-forming fungi such as Cortinarius and Tricholoma species have a high C:N demand (Trudell and Edmonds 2004) and are characteristically protein-users (Lilleskov et al. 2011), which may supply their requirement for both N and C. Host plants may also be able to employ mechanisms such as greater root tip longevity (Hoeksema and Kummel 2003) or increased carbohydrate allocation (Kiers et al. 2011) to promote association with fungi providing greater quantities of N. Results of N-form preference studies of ECM mycelia in culture should, therefore, be supported with observations of the symbiosis, as N-form uptake and assimilation are affected by C availability (Quoreshi et al. 1995) and the relation between N and C metabolism (Scheromm et al. 1990). N-form uptake rates and proportions and assimilation by ECM supplied with C from their plant hosts can, therefore, differ from those of the fungi, alone (Turnbull et al. 1995).
The extraordinary ability of ECM fungi and their plant partners to fully exploit the wide range of available N sources found in soils demands further investigation. Ensuring the partnership with ECM fungi is appropriate to the soil conditions and the desired vegetation cover could make a major contribution to the success or failure of restoration efforts.
Part II: The ectomycorrhizal fungus × tree interaction and restoration
Following Frank’s first, formal description of ECM in 1885, almost 25 years of debate ensued over whether the association was beneficial or pathological (Trappe 1977). While the fungus may receive up to 22 % of total plant carbohydrate allocation (Hobbie 2006) and form a stronger sink for C than plant growth during the early establishment of the symbiosis (Menkis et al. 2011), there is strong evidence that ECM improve survival, establishment and growth of seedlings in forest plantations over the long term. Lists of the beneficial effects for the plant of ECM partnership include: improved uptake of water and nutrients, particularly nutrients with low diffusion rates or that occur in complex forms; increased root longevity and growth; protection of roots against drought, high temperatures, pathogens, heavy metals, alkalinity and salts; and improved soil aggregation (Davey 1990; Menkis et al. 2011).
Ectomycorrhizae and plant N uptake
Plants that form ECM can grow on a wide variety of site types (Dickie et al. 2013), but generally predominate in natural environments where N accumulates in organic surface horizons with low N availability that may limit plant growth alone, or in combination with P (Read 1991; Lambers et al. 2008). Wang and Qiu (2006) suggest that the plasticity of ECM evolution and the diversity of ECM fungal species reflect an opportunistic response of plants and fungi to environmental stress that has spurred diversification in both plant and fungal lineages. In northern temperate and boreal forests where available N limits plant growth (Millard 1996), symbiosis with ECM fungi allows plants greater access to soil N pools, and mycorrhizal effects on growth depend more on N than P gain (Corrêa et al. 2012). ECM fungi efficiently take up inorganic N and amino acids from soils and 87 % of species tested can produce protease or grow on protein as a pure N source, demonstrating their ability to break down organic N polymers and share the N with the plant partner (Talbot and Treseder 2010). As plants are also able to take up N in inorganic and organic forms, the diversity of extracellular enzymes released by ECM into the soil may be of particular benefit to plants when inorganic N is at low availability but organic N pools exist. With a sufficient inorganic N supply, ECM association may depress seedling growth due to the rapid growth and C sink of extraradical mycelium (Plassard et al. 2000). In conditions of limited inorganic N but sufficient organic N supply, however, mycorrhizal association can benefit seedling growth and N content (Abuzinadah and Read 1986b; Plassard et al. 2000).
N uptake by ECM fungi can benefit the plant partner as N concentrations in the tissues of mycorrhizal plants are often higher than plants without mycorrhizae (Finlay et al. 1988). Studies of the plant/fungal partnership have clearly demonstrated enhanced uptake of N by some ECM plants. Uptake and assimilation of 15N-labelled ammonium into a variety of amino acids, particularly glutamate/glutamine, aspartate/asparagine and alanine was observed in mycelium, root and shoot tissues of Scots pine seedlings, with 5–50 % of the amino acids in the shoots being labeled (Finlay et al. 1988). Using microelectrodes, Gobert and Plassard (2002) and Plassard et al. (2002) consistently showed higher rates of nitrate uptake in short roots of maritime pine colonized by Rhizopogon roseolus (Corda) Th. M. Fr. than in non-mycorrhizal roots, and we have found similar trends for ammonium uptake measured with microelectrodes in roots of Douglas-fir colonized by a range of ECM (Fig. 1). By contrast, maritime pine roots colonized by Hebeloma cylindrosporum Romagn. had lower rates of nitrate uptake than non-mycorrhizal roots, and seedlings associated with this ECM fungal species had poorer growth than non-mycorrhizal plants (Plassard et al. 2002). In field settings under much less controlled conditions, Engelmann spruce (Picea engelmanniiParry ex. Engelm.) seedlings colonized by a Wilcoxina sp. assimilated significantly more N than seedlings colonized by Cenococcum spp., regardless of whether the N was applied as nitrate, ammonium or aspartate, with non-mycorrhizal plants intermediate (Jones et al. 2009). In degraded gypsum soils, Aleppo pine (Pinus halepensis Mill.) seedlings inoculated with Suillius collinitus (Fr.) Kuntze had greater survival, height growth and N concentration than seedlings inoculated with Amanita avoidea (Bull.), while inoculation with Rhizopogon roseolus caused significant seedling mortality (Rinćon et al. 2007). These results indicate that differences in N uptake among ECM fungi can be greater than between ECM and non-mycorrhizal plants.
As described in Part I, inorganic N deposition has a negative effect on many ECM fungi, but is the overall result deleterious for tree growth? Can the loss of ECM fungal community diversity or abundance from N fertilization or deposition be compensated for by increased N availability? European and American studies indicate that long-term N deposition will negatively affect tree growth and survival, in part due to induced P deficiency (Braun et al. 2010). Increased levels of N can increase plant demand for P, and ironically, the alterations in ECM fungal partners could reduce the tree’s ability to obtain P from the soil. As an interesting aside, alders (Alnus spp.), N-fixing, ECM trees with abundant N provided by their symbiont, Frankia, have a species-poor but highly host-specific ECM fungal community with high organic P acquisition ability (Walker et al. 2014). Red alder (Alnus rubra Bong.) had only 25 % of the number of ECM fungal species found on sympatric Douglas-fir but the alder ECM fungal community had significantly higher P potential enzyme activities (Walker et al. 2014). Perhaps chronic N deposition on forests over the long term will select for ECM fungi with high P acquisition ability.
Forest fertilization, particularly with N and combinations of N and other essential elements, is being used increasingly in North American and Europe to stimulate tree growth for reforestation or restoration purposes (Jones et al. 2012). Fertilization differs from N deposition in that nutrient applications are episodic, and effects of fertilization on ECM appear to be related to the frequency of fertilizer application. Infrequent N fertilization (≥7 year intervals) had no effect on ECM colonization of Scots pine seedling roots (Arnebrant and Söderström 1992) or on ECM species or diversity of 24-year-old western hemlock (Tsuga heterophylla (Raf.) Sarg.) (Wright et al. 2009). When fertilization occurs annually, however, ECM fungal species composition or richness (Fransson et al. 2000; Jones et al. 2012), colonization or both (Berch et al. 2006) are negatively affected. In a meta-analysis of 23 ECM studies, N fertilization reduced the percentage of ECM root tips, overall, by 15 % (Treseder 2004). Again, the question can be asked, does the overall negative effect of repeated fertilization on many ECM fungi have negative consequences for tree growth? Based on a study of ECM enzymes in lodgepole pine stands subject to annual fertilization with N, P, K, S, Mg and B, Jones et al. (2012) concluded that shifts in species composition with fertilization did not reduce the ability of the ECM fungal community to degrade soil organic matter and cycle nutrients; thus fertilization-induced changes in the fungal community would likely not affect tree growth. Effects on tree growth may also depend on the N–C balance between soil, fungi and trees. Teste et al. (2012) found that thinning alleviated the negative effects of recent fertilization on ECM abundance and suggested that greater C acquisition and root exploration by the trees after thinning maintained ECM abundance. When soil N is relatively abundant and tree-derived C is limited, fertilization may have negative effects on the fungal community, whereas fertilization effects may be mitigated if the tree has surplus C on a low-N site (Berch et al. 2006, 2009). These studies suggest that initial or infrequent fertilizer application to ensure the survival of seedlings on restoration sites would not pose a long-term impediment to the succession of ECM fungi.
Part II: The ectomycorrhizal fungus × tree interaction and restoration
Following Frank’s first, formal description of ECM in 1885, almost 25 years of debate ensued over whether the association was beneficial or pathological (Trappe 1977). While the fungus may receive up to 22 % of total plant carbohydrate allocation (Hobbie 2006) and form a stronger sink for C than plant growth during the early establishment of the symbiosis (Menkis et al. 2011), there is strong evidence that ECM improve survival, establishment and growth of seedlings in forest plantations over the long term. Lists of the beneficial effects for the plant of ECM partnership include: improved uptake of water and nutrients, particularly nutrients with low diffusion rates or that occur in complex forms; increased root longevity and growth; protection of roots against drought, high temperatures, pathogens, heavy metals, alkalinity and salts; and improved soil aggregation (Davey 1990; Menkis et al. 2011).
Ectomycorrhizae and plant N uptake
Plants that form ECM can grow on a wide variety of site types (Dickie et al. 2013), but generally predominate in natural environments where N accumulates in organic surface horizons with low N availability that may limit plant growth alone, or in combination with P (Read 1991; Lambers et al. 2008). Wang and Qiu (2006) suggest that the plasticity of ECM evolution and the diversity of ECM fungal species reflect an opportunistic response of plants and fungi to environmental stress that has spurred diversification in both plant and fungal lineages. In northern temperate and boreal forests where available N limits plant growth (Millard 1996), symbiosis with ECM fungi allows plants greater access to soil N pools, and mycorrhizal effects on growth depend more on N than P gain (Corrêa et al. 2012). ECM fungi efficiently take up inorganic N and amino acids from soils and 87 % of species tested can produce protease or grow on protein as a pure N source, demonstrating their ability to break down organic N polymers and share the N with the plant partner (Talbot and Treseder 2010). As plants are also able to take up N in inorganic and organic forms, the diversity of extracellular enzymes released by ECM into the soil may be of particular benefit to plants when inorganic N is at low availability but organic N pools exist. With a sufficient inorganic N supply, ECM association may depress seedling growth due to the rapid growth and C sink of extraradical mycelium (Plassard et al. 2000). In conditions of limited inorganic N but sufficient organic N supply, however, mycorrhizal association can benefit seedling growth and N content (Abuzinadah and Read 1986b; Plassard et al. 2000).
N uptake by ECM fungi can benefit the plant partner as N concentrations in the tissues of mycorrhizal plants are often higher than plants without mycorrhizae (Finlay et al. 1988). Studies of the plant/fungal partnership have clearly demonstrated enhanced uptake of N by some ECM plants. Uptake and assimilation of 15N-labelled ammonium into a variety of amino acids, particularly glutamate/glutamine, aspartate/asparagine and alanine was observed in mycelium, root and shoot tissues of Scots pine seedlings, with 5–50 % of the amino acids in the shoots being labeled (Finlay et al. 1988). Using microelectrodes, Gobert and Plassard (2002) and Plassard et al. (2002) consistently showed higher rates of nitrate uptake in short roots of maritime pine colonized by Rhizopogon roseolus (Corda) Th. M. Fr. than in non-mycorrhizal roots, and we have found similar trends for ammonium uptake measured with microelectrodes in roots of Douglas-fir colonized by a range of ECM (Fig. 1). By contrast, maritime pine roots colonized by Hebeloma cylindrosporum Romagn. had lower rates of nitrate uptake than non-mycorrhizal roots, and seedlings associated with this ECM fungal species had poorer growth than non-mycorrhizal plants (Plassard et al. 2002). In field settings under much less controlled conditions, Engelmann spruce (Picea engelmanniiParry ex. Engelm.) seedlings colonized by a Wilcoxina sp. assimilated significantly more N than seedlings colonized by Cenococcum spp., regardless of whether the N was applied as nitrate, ammonium or aspartate, with non-mycorrhizal plants intermediate (Jones et al. 2009). In degraded gypsum soils, Aleppo pine (Pinus halepensis Mill.) seedlings inoculated with Suillius collinitus (Fr.) Kuntze had greater survival, height growth and N concentration than seedlings inoculated with Amanita avoidea (Bull.), while inoculation with Rhizopogon roseolus caused significant seedling mortality (Rinćon et al. 2007). These results indicate that differences in N uptake among ECM fungi can be greater than between ECM and non-mycorrhizal plants.
As described in Part I, inorganic N deposition has a negative effect on many ECM fungi, but is the overall result deleterious for tree growth? Can the loss of ECM fungal community diversity or abundance from N fertilization or deposition be compensated for by increased N availability? European and American studies indicate that long-term N deposition will negatively affect tree growth and survival, in part due to induced P deficiency (Braun et al. 2010). Increased levels of N can increase plant demand for P, and ironically, the alterations in ECM fungal partners could reduce the tree’s ability to obtain P from the soil. As an interesting aside, alders (Alnus spp.), N-fixing, ECM trees with abundant N provided by their symbiont, Frankia, have a species-poor but highly host-specific ECM fungal community with high organic P acquisition ability (Walker et al. 2014). Red alder (Alnus rubra Bong.) had only 25 % of the number of ECM fungal species found on sympatric Douglas-fir but the alder ECM fungal community had significantly higher P potential enzyme activities (Walker et al. 2014). Perhaps chronic N deposition on forests over the long term will select for ECM fungi with high P acquisition ability.
Forest fertilization, particularly with N and combinations of N and other essential elements, is being used increasingly in North American and Europe to stimulate tree growth for reforestation or restoration purposes (Jones et al. 2012). Fertilization differs from N deposition in that nutrient applications are episodic, and effects of fertilization on ECM appear to be related to the frequency of fertilizer application. Infrequent N fertilization (≥7 year intervals) had no effect on ECM colonization of Scots pine seedling roots (Arnebrant and Söderström 1992) or on ECM species or diversity of 24-year-old western hemlock (Tsuga heterophylla (Raf.) Sarg.) (Wright et al. 2009). When fertilization occurs annually, however, ECM fungal species composition or richness (Fransson et al. 2000; Jones et al. 2012), colonization or both (Berch et al. 2006) are negatively affected. In a meta-analysis of 23 ECM studies, N fertilization reduced the percentage of ECM root tips, overall, by 15 % (Treseder 2004). Again, the question can be asked, does the overall negative effect of repeated fertilization on many ECM fungi have negative consequences for tree growth? Based on a study of ECM enzymes in lodgepole pine stands subject to annual fertilization with N, P, K, S, Mg and B, Jones et al. (2012) concluded that shifts in species composition with fertilization did not reduce the ability of the ECM fungal community to degrade soil organic matter and cycle nutrients; thus fertilization-induced changes in the fungal community would likely not affect tree growth. Effects on tree growth may also depend on the N–C balance between soil, fungi and trees. Teste et al. (2012) found that thinning alleviated the negative effects of recent fertilization on ECM abundance and suggested that greater C acquisition and root exploration by the trees after thinning maintained ECM abundance. When soil N is relatively abundant and tree-derived C is limited, fertilization may have negative effects on the fungal community, whereas fertilization effects may be mitigated if the tree has surplus C on a low-N site (Berch et al. 2006, 2009). These studies suggest that initial or infrequent fertilizer application to ensure the survival of seedlings on restoration sites would not pose a long-term impediment to the succession of ECM fungi.
Benefits of ECM inoculation depend on disturbance regime
The ability of many ECM fungi to improve access to and uptake of limited soil N is clear, however debate continues over where in the spectrum of forest regeneration/restoration, nursery inoculation, as opposed natural ECM colonization after planting, is required to achieve optimal growth of tree seedlings.
Degraded and extreme sites
Nursery inoculation with ECM-forming fungi is demonstrably beneficial when tree seedlings are planted in soils without suitable ECM fungal inoculum. A need for mycorrhizal inoculation of seedlings in the nursery became evident in the early twentith century when pines such as slash pine (Pinus elliottii Engelm.), patula pine (P. patula Schiede ex Schltdl. and Cham.) and radiata pine (P. radiata D.Don) were grown in nurseries south of the equator, in locations where no ECM fungal hosts, and therefore no ECM fungal inoculum, were present (Trappe 1977). By the mid-twentith century, inoculation was recognized as also necessary for plantations in steppe and prairie soils lacking ECM inoculum. Research continues to show the benefits of nursery ECM inoculation in restoration of degraded sites such as oil sands reclamation sites (Onwuchekwa et al. 2014). In a meta-analysis of field experiments on restoration of degraded drylands, inoculation with mycorrhizal fungi in the nursery, and tree shelters were the most effective treatments for enhancing survival and growth of planted seedlings (Piñeiro et al. 2013). An additional important but often over-looked benefit is the contribution of mycorrhizae to improving soil structure by increasing soil aggregates (Miller and Jastrow 1992). On severely degraded sites where ECM diversity has been lost, inoculation with a diverse suite of fungal species may increase the probability of a positive effect. (Sousa et al. 2014a).
Inoculation with ECM fungi has also been shown to improve survival and growth of seedlings on arid sites (Turgeman et al. 2011; Sebastiana et al. 2013) where reduced nutrient availability compounds the effects of water stress. Nursery inoculation of cork oak (Quercus suber L.) with Pisolithus tinctorius (Mich.: Pers.) Coker & Couch improved N content, biomass, leaf area, photosynthetic capacity and water use efficiency in the nursery, leading to increased survival and growth in the first year after planting in a dry, Mediterranean woodland (Sebastiana et al. 2013). In a mesic environment, inoculation with Paxillus involutus (Batsch: Fr.) Fr. stimulated the growth of young oak saplings, but only in dry years (Garbaye and Churin 1997). Many mechanisms have been proposed for the positive effect of ECM on dry sites; however, in a recent review, Lehto and Zwiazek (2011) concluded that the most important function of mycorrhizae during drought may be to facilitate nutrient, including N, acquisition.
Metal-contaminated sites
Mine tailings and soils adjacent to smelters can be challenging to revegetate because they are contaminated by heavy metals. Other soils can have naturally elevated concentrations of metals in soil solution because of low soil pH [aluminum (Al), iron (Fe), manganese (Mn)] or enriched parent materials [nickel (Ni), chromium (Cr) and others on ultramafic deposits] (Jourand et al. 2014). Trees may be planted on these contaminated sites to re-establish a functioning ecosystem or to assist in phytoremediation through bioaccumulation of metals in plant tissues. Excess levels of nutrient [copper (Cu), Mn, Zn, Ni] and non-nutrient [Al, mercury (Hg), cadmium (Cd), cobalt (Co), lead (Pb)] heavy metals impede plant growth by interfering with metabolism, including nutrient uptake; increasing mutation rates and DNA damage; and interfering with mitosis, resulting in stunted growth, especially in roots (Luo et al. 2014). An ECM fungus with a greater tolerance to metals than its host has the potential to increase the survival and growth of that host on a contaminated site. This is, in part, because metal ions must pass through the ECM fungal mantle in order to reach root tissues.
There are several mechanisms by which ECM fungi can reduce accumulation of metals in root and shoot tissues (Luo et al. 2014). Tolerant fungal strains may pump metal ions out of hyphae at faster rates than sensitive strains (Ruytinx et al. 2013; Majorel et al. 2014), or may chelate metal ions extracellularly, which reduces uptake (Meharg 2003; Johansson et al. 2008). Concentrations of metal atoms in the fungal cytosol can be reduced by sequestering metals in the endoplasmic reticulum (Blaudez and Chalot 2011) or vacuole (Luo et al. 2014). Alternatively, metals can remain in the cytoplasm, but be detoxified by binding to cysteine-rich proteins called metallothioneins (Bellion et al. 2007; Ramesh et al. 2009). Surprisingly, however, reduced accumulation of metals in the plant is only poorly correlated with ECM amelioration of plant performance under contaminated conditions (Jentschke and Goldbold 2000; Luo et al. 2014). In some cases, metal accumulation by the plant remains the same, but concentrations are diluted when growth rates increase as a consequence of better nutrition, overall (Jentschke and Goldbold 2000). Furthermore, colonization by ECM fungi appears to trigger more detoxification of metals within plant tissues, perhaps through increased expression of metal transporters associated with sub-cellular sequestration or amelioration of oxidative stress (Ma et al. 2014).
There is increasing interest in using ECM fungi as part of phytoremediation of metal-contaminated sites, where there is an interest in removal of metals from soils by plants (Rajkumar et al. 2012). Willow, aspen, popular and birch are popular choices for phytoextraction of toxic metals because they are fast-growing. Inoculation with specific isolates of ECM fungi can increase metal accumulation from metal-contaminated substrates either through growth stimulation or increased uptake of the metal (e.g. Fernandez et al. 2008; Hrynkiewicz and Baum 2013).
Whether a plant growing in a contaminated substrate will benefit from colonization by an ECM fungus depends on the fungus/plant/metal combination (Jones and Hutchinson 1986; Baum et al. 2006; Aggangan and Jones 2012). Ectomycorrhizal fungi can vary in their degree of metal tolerance (Jones and Hutchinson 1986) and tree species differ in their response to inoculation with a given ECM fungus in metal-contaminated soils (Mrnka et al. 2012). Interactions between ECM fungi and plants can vary even at the genotype level: Sousa et al. (2014b) found a plant genotype X ECM fungal species interaction with respect to Cd accumulation, when Scots pine seedlings were inoculated with Suillus bovinus or Rhizopogon roseolus. Because of the specificity of the amelioration effects of ECM fungi, considerable testing should be done prior to the instigation of any inoculation program.
Not all fungal isolates from a contaminated site will be tolerant to the metals found on that site (Majorel et al. 2012). A standard technique for screening tolerant isolates is to grow them in pure culture in liquid or solidified nutrient media amended with a range of concentrations of toxic metals. Although this technique has been successful in detecting some ECM fungi that ameliorate metal tolerance in their plant partners (Adriaensen et al. 2006), it is not always effective. For example, inoculation of wattle (Acacia spirorbis Labill.) and blue gum (Eucalyptus globulus Labill.) with a metal-sensitive isolate of Pisolithus albus(Cooke and Massee) stimulated shoot growth on an ultramafic soil high in Co, Cr, Fe, Mn and Ni more than several metal-tolerant isolates of the same fungal species (Jourand et al. 2014). Jones and Hutchinson found that fungal isolates with very low tolerance to Ni in pure culture (Jones and Hutchinson 1988a) produced the most biomass and provided the most protection for plants against Ni toxicity (Jones and Hutchinson 1988b). These results confirm the importance of testing a wide range of combinations of plant genotypes and fungal isolates in symbiosis in realistic substrates when determining the efficacy of growth enhancement by ECM fungi on metal-contaminated sites.
Clearcuts
In a clearcut forest stand, most ECM roots die within 2 years of harvest, reducing the source of inoculum (reviewed in Wiensczyk et al. 2002). Similarly, stockpiled topsoil shows a significant reduction in mycorrhizal inoculation potential after 3 years (Miller and Jastrow 1992). Nursery inoculation with one ECM fungal species can increase, decrease or not affect seedling survival and/or growth after outplanting, but these inconsistent results may depend, in part, on the fungal species used (Kropp and Langlois 1990) and the time since forest harvest (reviewed in Jones et al. 2003). Nursery cultural techniques that encourage natural ECM colonization in the nursery, such as reduced nutrient inputs and the use of well-drained, coarse textured substrates, will result in seedling colonization by pioneer ECM species often found in clearcuts [Thelephora terrestris Ehrh., Amphinema byssoides (Pers.) J. Erikss.] (Quoreshi et al. 2009). Encouraging colonization by the most appropriate ECM fungal species may be more important than the number of species applied in inoculum. Kranabetter (2004) found a low diversity suite of pioneer ECM fungal species (average 2.7 morphotypes per seedling) resulted in greater spruce seedling N content and height growth on a clearcut than a higher diversity suite of mature forest ECM fungal species (average 5.3 morphotypes per seedling), suggesting that the ECM communities from disturbed soils are well adapted to the soil conditions of recent clearcuts.
Ectomycorrhizae formed on tree seedling roots in the nursery typically decrease in abundance over time after planting in clearcuts, but nursery fungi can persist for 8 (Jones et al. 2003) to 10 years (Selosse et al. 1998). Fungi already present on a root system may colonize new roots more easily than other ECM fungi, thus healthy growth of nursery fungi can suppress colonization by indigenous fungi for some time (Jones et al. 2003).
Partial cuts, patch cuts and edges
In partial cuts, patch cuts or near the forest edge of clearcuts, seedlings may benefit directly and indirectly from nearby mature trees, including from mycorrhizal inoculum (Baker et al. 2013), and nursery inoculation likely provides no additional benefit to seedling survival or nutrition. Many studies have documented increased ECM fungal colonization and diversity in naturally regenerated and planted seedlings located closer to the forested edge of clearcuts (Jones et al. 2003); however, this does not necessarily translate into improved seedling nutrition or increased seedling growth (Hagerman et al. 1999). At forest edges, a relatively narrow zone of forest influence of 10 m or less has been documented for mycorrhizal colonization (Jones et al. 2008; Outerbridge and Trofymow 2009; Baker et al. 2013) as ECM fungal species richness is related to root density (Luoma et al. 2006). Greater green tree retention in partial cuts can increase fungal richness and mycorrhizal colonization distance from the forest edge in planted seedlings (Outerbridge and Trofymow 2009). Kranabetter (1999) found 38 % more types of ECM fungi in naturally regenerated paper birch seedlings growing within the rooting zone of mature trees in clearcuts, compared to seedlings outside the mature rooting zone. In variable retention harvesting, ECM fungal community composition is simplified and species richness and diversity are reduced with increasing distance from mature trees (Teste et al. 2009). ECM fungal exploration types are correlated with distance from forest edges, where long-distance exploration type-fungi, with smooth mantles and few but highly differentiated rhizomorphs (Agerer 2001), are more prevalent farther from the forest edge in low root density regions, while short-distance exploration types, with a voluminous envelope of emanating hyphae but no rhizomorphs (Agerer 2001), are more common near forest edges in areas of high root density (Peay et al. 2011).
In reforestation of small patches, planted seedlings may link to existing plants through mycorrhizal networks, ECM mycelia that connect the roots of several host plants. Mycorrhizal networks have been found in all major terrestrial ecosystems and often link overstory and understory species (Simard et al. 2012). These linkages may facilitate transfer of C, nutrients, water or defense compounds among trees (reviewed in Simard et al. 2012; Bingham and Simard 2013). Resource sharing through mycorrhizal networks is suggested to improve seedling establishment, particularly in dry or infertile conditions. In contrast to the view that mycorrhizal networks might facilitate nutrient sharing among plants (van der Heijden and Horton 2009), Franklin et al. (2014) suggest such networks enhance competition for N and C among trees and fungi. N retention by ECM fungi in forest stands may negatively affect plant productivity under N-limiting conditions due to reduced N transfer from the fungus, rather than costly C allocation by the plant (Corrêa et al. 2012). At low N availability, such as in boreal forest soils, ECM fungi can retain N for their own growth (Näsholm et al. 2013), possibly exacerbating soil N limitation by N immobilization in fungal biomass. High colonization rates are clearly maintained, however, which may be due to the multiple fungal and plant partnerships (Franklin et al. 2014), the diverse benefits of the ECM symbiosis, or both.
Effects of ECM diversity
Interspecific diversity
It is generally assumed that colonization by native ECM fungi will be superior to nursery ECM “contaminants” such as Thelephora terrestris (e.g. Perry et al. 1989) and that a more diverse mycorrhizal community will result in greater tree productivity than one or two ECM fungal species, but these hypotheses are difficult to test and results may differ depending on the conditions of the experiment (Jonsson et al. 2001; Kranabetter 2004). Furthermore, both T. terrestris and Wilcoxina mikolae Yang & Korf, another common nursery ECM fungus, are effective at stimulating nutrient uptake and growth of young seedlings relative to non-mycorrhizal plants (Jones et al. 1990, 2009). A more diverse ECM fungal community could provide an advantage to the plant through greater functional diversity and access to a greater diversity of N forms (Finlay et al. 1992), P pools (Velmala et al. 2014), and/or organic matter (Jones et al. 2010), potentially reducing below-ground competition (Perry et al. 1989). Alternatively, exposure to more ECM fungal species could increase the likelihood of association with a critical fungal species. Evidence exists for both scenarios. Baxter and Dighton (2001) found that diversity, per se, was an advantage rather than the presence of a critical species, as biomass allocation to roots of grey birch seedlings (Betula populifoliaMarsh.) increased with fungal diversity (1, 2 or 4 ECM fungal species), along with a small increase in shoot N concentration and an increase in P content. Likewise, a mix of eight ECM fungal species improved growth of European silver birch seedlings (Betula pendula Roth.) in low, but not high fertility soil, relative to two or four ECM species (Jonsson et al. 2001). By contrast, seedlings of some conifers appear to perform better with fewer fungal partners, and Velmala et al. (2013) suggested that nutrient acquisition of young trees may benefit most from colonization with only one or two ECM fungal species. Pairs of ECM fungal species increased Scots pine growth in high fertility soil, relative to colonization by eight ECM fungi (Jonsson et al. 2001), and shoot growth of drought-stressed Scots pine seedlings was enhanced in the presence of Suillus granulatus (L.) Roussel alone, but not by mixtures of two, three or four fungal species (Kipfer et al. 2012). N concentration of needles of Norway spruce cuttings colonized by up to five ECM fungal species was also found to be negatively correlated with ECM richness (Velmala et al. 2013).
Another potential advantage of high fungal species richness in ECM associations is insurance against changing environmental conditions (Pena et al. 2010; O’Hanlon 2012). Pena et al. (2010) suggest plants make a small investment of recent photoassimilate in many rare ECM fungal species, but when carbon is limited, only the most predominant fungal species are supported. When European beech trees were girdled, preventing supply of new photoassimilate to roots, ECM species richness was reduced by roughly 50 % but the ECM fungal species lost had colonized only 8 % of root tips (Pena et al. 2010). These rare species may be insurance for nutrient uptake if environmental conditions change.
Despite potential benefits of high ECM fungal species richness in the long term, successful forest restoration appears not to require a high diversity of ECM fungi at establishment, even on harsh sites (relatively cold or dry) or after severe disturbances such as wildfire. Natural ECM colonization of seedlings may be relatively slow on difficult sites, however. For example, in dry Douglas-fir forests, only 20 or 50 % of seedling roots were colonized by ECM fungi after 1 year on burned or clearcut sites, respectively; and 80–100 % of mycorrhizas were formed by Wilcoxina spp. (Barker et al. 2013). Comparable rates of colonization were observed in lodgepole pine in the first year after wildfire (Miller et al. 1998). Similarly, roots of Engelmann spruce planted in recent clearcuts of cold, high elevation forests averaged 20–30 % colonization by one ECM fungal species (maximum three) after a 13 week growing season (Hagerman et al. 1999). These studies indicate that nursery inoculation with a few appropriate ECM fungi will be sufficient to promote seedling establishment on degraded sites.
Intraspecific diversity
Plant and fungal species richness, evenness and species combinations have significant effects on ecosystem functioning, but few studies of ECM have considered the importance of variation among individuals within species (Johnson et al. 2012). Within-species variation exists in non-mycorrhizal tree root architecture and morphology, N uptake rates and N form preference (Hawkins 2007; Miller and Hawkins 2007; Velmala et al. 2013), and intra-specific variation in ECM colonization and host response has been shown in trees (Tagu et al. 2001; Karst et al. 2009). Intra-specific functional diversity in ECM fungal species for enzyme activity, N form preference and N uptake (Scheromm et al. 1990; Guidot et al. 2005) also indicate variation within fungal species in the use of N sources. Great potential exists for selection of plant genotypes, fungal genotypes or complementary plant genotype × fungal genotype interactions (reviewed in Johnson et al. 2012) to improve N uptake and the success of forest regeneration and restoration.
Mycorrhizal fungal strains may be adapted to or selected by the available N forms in their native soils (Chalot and Plassard 2011), and the degree of variation in growth rates among fungal genotypes has been shown to be as great as among fungal species under different substrate C:N ratios (Johnson et al. 2012). These genetic differences in growth rates were substantially greater than growth differences effected by a fourfold change in C:N ratio (Johnson et al. 2012). A mix of genotypes may also be used to advantage, as growth of fungal cultures was 50 % greater in mixtures of eight genotypes than with a single genotype (Wilkinson et al. 2010).
Variation can also occur within tree species in their ability to form ECM symbioses and respond to ECM association. The provenance of 2-year-old Scots pine influenced ECM fungal species richness, and provenance survival and N status was positively linked with the abundance of Suillus species (organic N-adapted) and negatively correlated with abundance of Wilcoxina species (inorganic N-adapted) (Leski et al. 2010). Northern European Scots pine provenances have earlier growth cessation and greater below-ground C allocation than central populations, which may support the greater C demands of thick-mantled ECM fungi such as Suillus species (Leski et al. 2010). Tree host genotype also played a significant role in the formation of ECM and in shaping the ECM fungal community structure in young Norway spruce clones, through genotypic differences in short root formation and growth (Velmala et al. 2014). Tall Norway spruce clones were found to have higher ECM diversity than short clones at 11 years of age (Korkama et al. 2006). Similarly, fast-growing, mature trees of pinyon pine (Pinus edulis Engelm.) were associated with more stable ECM fungal partners, considered superior mutualists, than slow-growing trees (Gehring et al. 2014).
By contrast, other studies show slow-growing tree families to benefit more from association with ECM fungi. For instance, association with Paxillus involutus was relatively more beneficial for slow-growing families of Norway spruce than for fast-growing families (Boukcim and Plassard 2003). Slow-growing families of Norway spruce also exhibited a stronger positive shoot growth response when colonized with a variety of ECM fungi than fast-growing families with no difference in ECM fungal diversity (Velmala et al. 2014). These authors suggest that root architecture may have a significant effect on ECM species diversity and segregation in the field, however, and that fast-growing trees with a spreading root system, lower fine root density and greater below-ground allocation will recruit more ECM species, leading to greater functional diversity and better plant growth in the long term (Velmala et al. 2014). Investigation is required in a wider range of tree species to understand this “chicken and egg” question: are faster growth rates a cause or an effect of the particular suite of ECM fungal symbionts?
Plant × fungal genotype interactions are evident in enzyme production. In Populus hybrids, the level of enzymes secreted by ECM root tips is under the genetic control of the host (Courty et al. 2011). Significant differences were found between poplar genotypes in the activities of most ECM enzymes measured, whereas there were no differences in enzyme production of non-mycorrhizal root tips (Courty et al. 2011). Given the intraspecific variation in a wide range of traits of ECM fungi and trees, there appears to be potential to improve the success of forest restoration by selecting appropriate genetic strains of both plants and fungi in ECM symbioses.
Conclusion
When restoring or regenerating forests, mycorrhizal associations can benefit tree seedling survival and growth after planting, particularly on drought-prone, metal-contaminated, cold or N-poor sites. If seedlings available for planting are non-mycorrhizal, inoculation with mycorrhizal fungi in the nursery should be considered to boost seedling establishment after planting, especially on degraded sites. Alteration of nursery practices in ways that encourage spontaneous colonization by early successional ECM fungi in the nursery should also be considered. In situations where restoration of the full ecosystem is desired, planting of a representative mix of both ECM and AM native plants may speed ecosystem recovery. For ECM plants, inoculation with one or two early succession ECM fungal species may be all that is required, although some sacrifice of early shoot growth may be needed to establish the association. Local field trials may be necessary to determine the most appropriate fungal isolate for the tree species (or even plant genotype, if possible) and the limitations of the restoration site. Proteolytic ECM fungal species are suitable for cold temperate, boreal or alpine soils where relative availability of organic N is high; however some of these “protein fungi” (Abuzinadah and Read 1986a) are notoriously difficult to culture. Nitrophilic species may be preferred when inorganic N comprises a greater proportion of the soil N pool. Fast-growing tree genotypes with significant biomass allocation to roots and spreading root systems are likely to provide the best return in absolute growth from ECM association, but slow-growing genotypes may derive a greater proportional benefit.
Lessons from ecological studies of forest disturbance suggest that ECM communities need not be diverse in early stages of succession. The fungal community is expected to become increasingly rich with stand development, assuming sources of inoculum exist relatively close by, perhaps within 1 km (Peay et al. 2010). For ECM fungi, successful restoration would entail a recovery in species diversity during stand development, including many dozen species of fungi appropriate for tree host, soil and climatic conditions (Visser 1995; Twieg et al. 2007). While our knowledge of the ecology of ECM fungal species is inadequate to predict what ECM community will arise on a restored site over time, the many hundreds of ECM fungal species in natural ecosystems are wonderfully specialized and adapted to the nuances of soil fertility, encompassing the entire range from infertile to highly productive. Only extremely harsh conditions, such as high soil alkalinity (Soudzilovskaia et al. 2015), soil water saturation, heavy metal contamination or lack of suitable hosts might inhibit ECM fungi recolonization of a disturbed site over time.
Although ECM fungi are a key component of healthy forest ecosystems, they are not easily managed or manipulated. Many are difficult to culture and we know of no instances where a broadly diverse ECM community has been created artificially. This may be the biggest challenge to full ecological restoration of forests on widely disturbed landscapes with little natural woodland to supply fungal inoculum. Consequently, ongoing monitoring of ECM communities is worthwhile to assess the long-term ecological recovery of restored sites, in addition to short-term measures of tree seedling survival and growth.
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