Can ectomycorrhizal fungi circumvent the nitrogen mineralization for plant nutrition in temperate forest ecosystems?

Soil Biology & Biochemistry 43 (2011) 1109e1117

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Review

Can ectomycorrhizal fungi circumvent the nitrogen mineralization for plant nutrition in temperate forest ecosystems? Tiehang Wu* Department of Horticulture, The Pennsylvania State University, 103 Tyson Building, University Park, PA 16802, USA

a r t i c l e i n f o

a b s t r a c t

Article history: Received 5 April 2010 Received in revised form 1 February 2011 Accepted 2 February 2011 Available online 18 February 2011

Nitrogen (N) limits plant growth in many forest ecosystems. The largest N pool in the plantesoil system is typically organic, contained primarily within the living plants and in the humus and litter layers of the soil. Understanding the pathways by which plants obtain N is a priority for clarifying N cycling processes in forest ecosystems. In this review, the interactions between saprotrophic microorganisms and ectomycorrhizal fungi in N nutrition with a focus on the ability of ectomycorrhizal fungi to circumvent N mineralization for the nutrition of plants in forest ecosystems will be discussed. Traditionally, it is believed that in order for plants to fulfill their N requirements, they primarily utilize ammonium (NHþ 4) and nitrate (NO 3 ). In temperate forest ecosystems, many woody plants form ectomycorrhizas which significantly improves phosphorus (P) and N acquisition by plants. Under laboratory conditions, ectomycorrhizal fungi have also been proven to be able to obtain N from organic sources such as protein. It was thus proposed that ectomycorrhizal fungi potentially circumvent the standard N cycle involving N mineralization by saprotrophic microorganisms. However, in many forest ecosystems the majority of the proteins in the forest floor form complexes with polyphenols. Direct access of N by ectomycorrhizal fungi from a polyphenoleprotein complex may be limited. Ectomycorrhizal fungi may depend on saprotrophic microorganisms to liberate organic N sources from polyphenol complexes. Thus, interactions between saprotrophic microorganisms and ectomycorrhizal fungi are likely to be essential in the cycling of N within temperate forest ecosystems. Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: Saprotrophic microorganisms Ectomycorrhizal fungi Organic nitrogen (N) N mineralization N economy

1. Introduction Nitrogen (N) is a limiting resource for plant growth in many temperate forests (Gosz, 1981; Aber et al., 1989; Stump and Binkley, 1992). Plants acquire N from inorganic forms such as ammonium  (NHþ 4 ) and nitrate (NO3 ). However, some evidence indicates that under laboratory conditions ectomycorrhizal fungi can assist plants to obtain N from organic sources as well (Melin and Nilsson, 1953; Abuzinadah and Read, 1986, 1989a,b; Abuzinadah et al., 1986; Finlay et al., 1992; Turnbull et al., 1995). Moreover, some plant species, especially those from the arctic, can directly absorb amino acids (Millar and Schmidt, 1965; Chapin et al., 1993; Kielland, 1994; Schimel and Chapin, 1996; Näsholm et al., 1998). Thus, Chapin (1995) suggested that plants may “short-circuit” the traditional N mineralization pathway. Under field conditions for temperate species, however, the ability of plants to obtain organic N is not clear. Most of the organic N in the

* Department of Cell Biology, Microbiology and Molecular Biology, University of South Florida, 4202 East Fowler Avenue, Tampa, FL 33620, USA. Tel.: þ1 813 974 8967; fax: þ1 813 974 1614. E-mail address: [email protected]. 0038-0717/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2011.02.003

forest floor may not occur as free protein or amino acid, but may be bound into polyphenoleprotein complexes (Northup et al., 1995a,b; Qualls et al., 1991). Many ectomycorrhizal fungi lack the necessary enzymes to obtain N from this complex (Bending and Read, 1996b; Wu et al., 2005). Therefore, understanding the mechanisms by which ectomycorrhizal fungi utilize organic N complexed by polyphenolic substances remains a challenge to our understanding of N cycling. Saprotrophic microorganisms exist in the organic layers of all forest floors and may be of great importance to the N economies of ectomycorrhizal fungi and their hosts (Cairney and Meharg, 2002; Rousk and Nadkarni, 2009; Wu et al., 2005). This review will be stressed on the interactions between saprotrophic microorganisms and ectomycorrhizal fungi in temperate forest ecosystems and focused on the ability of ectomycorrhizal fungi to obtain N from organic forms by circumventing the process of N mineralization. 2. Nitrogen nutrition in forest ecosystems 2.1. Nitrogen as the limiting nutrient in temperate forest ecosystems Nitrogen may limit plant growth in many temperate forest ecosystems (Gosz, 1981; Aber et al., 1989; Stump and Binkley, 1992).

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This has been established primarily by recording positive growth responses to N fertilization (Mitchell and Chandler, 1939; Aber et al., 1989; Chappell et al., 1991; Binkley et al., 1995a, 1995b; Kiefer and Fenn, 1997; Wu et al., 2005). Some ecosystems that have accumulated nitrogen from heavy atmospheric deposition, however, are apparently not N-limited (Aber et al., 1989; Aber, 1992; Fisher and Binkley, 2000). The N-limitation of many ecosystems may be partly caused by significant N leaching, especially as nitrate (Vitousek et al., 1982). Significant leaching of dissolved organic N may also occur, and, in pristine, unpolluted ecosystems losses as dissolved organic N may exceed losses as nitrate (Perakis and Hedin, 2002a,b). 2.2. The traditional view of N mineralization In forest ecosystems the largest pool of N in the plantesoil system is typically organic, contained primarily within the living plants and in the humus and litter layers of the soil (Johnson, 1992). Litter is decomposed by a variety of microorganisms. Fungi are known to be the chief colonizers and decomposers of plant litter in temperate forests (Hudson, 1968; Swift et al., 1979), because it is within this group that there is the most widespread distribution of extracellular depolymerising enzymes such as cellulases or lignin degrading enzymes (Swift et al., 1979). Bacteria possess lesser abilities to produce extracellular, depolymerising enzymes. Therefore, fungi are particularly adapted to degrading plant materials and bacteria appear to be better suited to resources such as corpses of microorganisms and animals (Swift et al., 1979). The saprotrophic fungi are, therefore, important in N cycling in forest ecosystems. Saprotrophic fungi can mobilize complex organic N sources as simpler organic compounds (Bending and Read, 1996a,b; Dighton, 2007), and can mineralize organic N into inorganic N forms (Myrold, 1998), either of which can be absorbed by mycorrhizal fungi and plants (Bending and Read, 1997). In temperate forests, because most of the N is in the polyphenoleprotein complex (Northup et al., 1995a), the mobilization and/or mineralization of the complex by saprotrophic fungi is the key step in N cycling. Ammonifying microorganisms release ammonium from proteins and other organic nitrogen sources when the organic matter on which they live contains more N than is necessary for their growth (Sprent, 1987; Myrold, 1998). The conversion of organic N compounds to ammonium is mediated by a series of enzymes. Extracellular enzymes first break down organic N polymers (including protein, chitin, nucleic acids, etc.) into monomers such as amino acids, amino sugars and nucleotides. Monomers pass across the cell membrane and are further metabolized by intracellular enzymes into ammonium, which is released into the soil solution when the microorganism is not limited by N (Myrold, 1998). However, when substrate N concentrations are low, most of the N mobilized from organic N polymers is converted into microbial N, which can be mineralized only after sufficient C is lost by respiration (Swift et al., 1979). Therefore, substrate C:N ratios, in relation to the C and N requirement of the soil microorganisms, determine, in part, whether N will be immediately mineralized or immobilized (Boberg et al., 2010). 2.3. Utilization of organic N The inorganic N mineralized from organic N sources was traditionally thought to be the sole N source for plant growth. However, in recent years, organic N has been shown to be of direct importance to plant N nutrition (Chapin, 1995; Schimel and Bennett, 2004). Some plants directly absorb free amino acids (Miettinen, 1959; Millar and Schmidt, 1965; Tinsley, 1969). In fact,

some arctic species preferentially absorb amino acids over inorganic forms (Kielland, 1994; Näsholm et al., 1998; Chapin et al., 1993; Schimel and Chapin, 1996). Moreover, many forest tree species form ectomycorrhizas and more and more evidence suggests that ectomycorrhizal fungi directly facilitate the acquisition of N by their hosts from organic forms such as amino acids and proteins under laboratory conditions (Melin and Nilsson, 1953; Abuzinadah and Read, 1986, 1989a,b; Abuzinadah et al., 1986; Finlay et al., 1992; Turnbull et al., 1995; Lindahl and Taylor, 2004) and possibly in the field (Näsholm et al., 1998). Michelsen et al. (1996) reported that the difference of natural 15N:14N ratios expressed as d15N in different plants could be applied to assess the role of ericoid and ectomycorrhizal fungi for the organic N nutrition in fields. However, further studies suggested that this evidence is not enough to support the organic N utilization because many factors, such as losses of N as NH3 formed during hydrolysis of proteins or amino acids, affect the relative proportions of the different forms of potentially available N and the d15N values in plants (Lipson and Näsholm, 2001). The mechanisms by which ectomycorrhizal fungi facilitate the use of organic N by their hosts in the field, however, is not entirely clear. In temperate forests, because net aboveground production is often highly correlated with soil N mineralization rates (Pastor et al., 1984), mineralization of organic N still appears to be the primary mechanism by which many tree species obtain the majority of N (Tate et al., 1991). 2.4. Forms of N in the forest floor Nitrogen in the forest floor comes from either exogenous or endogenous sources. Atmospheric N deposition is an exogenous input. The living plants, animals and microorganisms represent endogenous sources of N (Vestgarden, 2001). In unfertilized and relatively unpolluted forest soils, plant and microbial growth depend primarily upon the recycling of endogenous N (Gorham et al., 1979; Sollins et al., 1980; Johnson, 1992; Perakis and Hedin, 2002a,b). Soil N can be characterized in several ways. It can be divided between organic and inorganic, dissolved and undissolved, or hydrolysable and unhydrolysable forms, based on the experimental methods used to extract N from soils. The method of extraction has a strong effect on soil N composition. For example, Abuarghub and Read (1988) indicated that comparisons of the amino acid composition of soils must be made with great caution because the procedures used for their extraction strongly influence the results. Non-gaseous inorganic N includes ammonium, nitrate and nitrite. Ammonium and nitrate frequently comprise the two largest pools of inorganic N. Ammonium is often the dominant inorganic N pool in forest ecosystems. Nitrate concentrations are normally smaller in mature forests because of small populations of nitrifying microorganisms and high concentration of alleochemical inhibition (Hart et al., 1994), because high concentrations of tannins and tannin derivatives inhibit nitrification (Rice and Pancholy, 1973), and because nitrate is readily absorbed by soil microbes and plants or leaches more readily from the soil compared to ammonium. In temperate forests, most of the soil N is organic. Wild (1988) estimated that only about 5e10% of total soil N is inorganic. Dissolved organic N (DON) has been identified as a key N pool in soileplant N cycling in forest ecosystems (Qualls et al., 1991). Inorganic N amounted to less than 5% of total dissolved N in a Douglas-fir forest floor (Griffiths et al., 1994). Organic forms of N include amino acids, amino sugars, proteins, peptides, chitin, other high-molecular weight polymers and unidentified organic N. (Wild, 1988). Sowden et al. (1977) determined the distribution of major N compounds in soils from different climatic and geological conditions including arctic, cool

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temperate, subtropical and tropical climates. Their results indicated that 33e42% of N occurred as amino acids, which were presumably hydrolyzed from the protein in the soil. During the extraction process, about 10% of the amino acid N was hydrolyzed to ammonia. Therefore, it was estimated that at least 40e50% of the total soil N was protein N (Schulten and Schnitzer, 1998). However, soil proteins are not necessarily free proteins, and they may not be soluble. Qualls et al. (1991) found that 94% of the dissolved N leaching through a deciduous forest soil was organic. Yu et al. (1994) also found that the dominant form of dissolved N in a coniferous forest soil was organic. Free proteins and amino acids constituted less than 5% of the total N when extracted at ambient soil solution pH (∼4.5). DON serves as an intermediate N pool between the inorganic N and insoluble organic N fractions during mineralization of organic matter (Appel and Mengel, 1990; Murphy et al., 2000). Microorganisms including ectomycorrhizal fungi and plants can also utilize directly the low molecular weight DON compounds as an N source (Murphy et al., 2000; Jones et al., 2005). Northup et al. (1995a) analyzed the composition of dissolved organic matter (DOC and DON) by the resin-exchange fractionation procedure and divided it into three different components: 1) the hydrophobic fraction which is dominated by high-molecular-mass phenolic acids including proteinetannin complexes; 2) the hydrophilic base fraction which is dominated by free proteins and amino acids; and 3) the hydrophilic acid fraction which is dominated by lowermolecular-mass ‘fulvic acids’. Research by Northup et al. (1995a) indicates that in pine forests most of the dissolved organic N is contained in the proteinetannin (polyphenol) complex fraction rather than as free amino acids or free proteins. Likewise, in an oak-dominated forest, 94% of the soluble N from the organic layer was organic, most of which was carried by humic substances and hydrophilic acids, not as free proteins or amino acids (Qualls et al., 1991). When polyphenolic compounds bind organic N, N mineralization is retarded (Handley, 1961; Hattenschwiler and Vitousek, 2000), as is N leaching (Northup et al., 1995a, 1998). The widely distributed polyphenolics in forest ecosystems may be important for the conservation of N in low fertility forest soils (Hattenschwiler and Vitousek, 2000). Plants and many ectomycorrhizal fungi are seem to be generally unable to access N from insouble polyphenoleprotein complex (Bending and Read, 1996a,b). Thus, Bending and Read (1996b) proposed that saprotrophic microorganisms first attack the complexes and further mobilize N and/or mineralize it before ectomycorrhizal fungi or plants can obtain it (Fig. 1).

Recalcitrant organic N Polyphenol-protein complex

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2.5. N turnover in the forest floor The pool of inorganic N is much smaller than that of organic N in the forest floor possible due to the rapid uptake of inorganic N by plants and microorganisms, or the rapid leaching of N as nitrate (Vitousek et al., 1982). The N mineralizationeimmobilization process generates the large annual flux through the N pool and supports a large fraction of the N required for plant growth (Berntson and Aber, 2000; Schmidt et al., 2007). Therefore, the N flux through the mineral N pool is far more important than the actual size of that pool. In non-fertilized forest soil, N mineralization provides the major N nutrition for plant growth, as it is through the processes of N mineralization that N contained within litter is mobilized. Gosz (1981) suggested that N-rich and N-poor sites produce litters of different quality therefore influence decomposition and mineralization of N differently. Vegetation on N-rich sites produces litter with high N concentrations and low concentrations of phenolics, leading to rapid mineralization of N. On N-poor sites, the litter possesses low N concentrations and high phenolic concentrations, and N mineralization is slow (Gosz, 1981). Root proliferation in patches of soil experiencing rapid N mineralization (Jackson and Bloom, 1990; Robinson et al., 1999; Hodge, 2004) suggests the importance of inorganic N to plants. However, we are beginning to learn about the relationship between organic N turnover and proliferation of ectomycorrhizal fungi in temperate forests (Bending and Read, 1995; Perez-Moreno and Read, 2000, 2001). 3. Ecotomycorrhizal fungi and N nutrition 3.1. The abundance of ectomycorrhizal host trees in temperate forest ecosystems Ectomycorrhizas are widely distributed in forest ecosystems. It is estimated that about 5000 fungal species and 2000 woody plant species form ectomycorrhizas (Molina et al., 1992; Lakhanpal, 2000). In coniferous forests of northern latitudes, more than 1000 species of ectomycorrhizal fungi associate with only a few plant species (Allen et al., 1995). Ectomycorrhizal fungi belong primarily to the Basidiomycota, but a few are in the Ascomycota or Zygomycota. Although more that 90% of terrestrial plant species form mycorrhizas, only about 3% are ectomycorrhizal (Meyer, 1973; Smith and Read, 1997), but these are frequently ecologically dominant species in the Pinaceae, Salicaceae, Betulaceae, Fagaceae and Myrtaceae (Meyer, 1973). Recently, more species have been

Dissolved organic N Saprotrophs

Free Protein, amino aiacids or

Saprotrophs

Inorganic N

1

polyphenol-protein complex

?

Ectomycorrhizal fungi

Plants Polyphenols Fig. 1. A proposed N cycling model in ectomycorrhizal forest systems (Modified from Chapin, 1995). Activities of saprotrophs are considered as tradition pathways for plants in obtaining N from the recalcitrant organic N (polyphenoleprotein complex). Ectomycorrhizal fungi obtaining N from organic forms in natural forest ecosystems was proposed, but without enough supporting evidence. Plants produce polyphenols and influence nutrient cycling by affecting organic matter degradation.

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found to form ectomycorrhizas, including some shrubs and a very small number of herbaceous angiosperms (Smith and Read, 1997). Some genera form ecto- as well as arbuscular mycorrhizas, such as Populus, Salix, and Eucalyptus (Lapeyrie and Chilvers, 1985; Chilvers et al., 1987; Lodge and Wentworth, 1990). Some plant species in the families traditionally thought to form only ectomycorrhizas, such as species in the Pinaceae and Fagaceae, have also been found to form arbuscular mycorrhizas (Cazares and Trappe, 1993; Smith et al., 1998; Dickie et al., 2001; Egerton-Warburton and Allen, 2001). 3.2. Role of ectomycorrhizal fungi in N nutrition of trees The beneficial effects of ectomycorrhizas are mainly due to enhanced nutrient acquisition by the host as a result of the fungal symbionts (Melin and Nilsson, 1953; Harley and Smith, 1983). The most important mechanisms by which mycorrhizal fungi increase nutrient uptake to their host plants include the great increase in the absorbing surface area, and the ability to exploit soil beyond the nutrient depletion zone that develops around the root (Smith and Read, 1997). That ectomycorrhizal fungi increase uptake of phosphorus has been widely demonstrated (Bolan, 1991; Read and Perez-Moreno, 2003). The role of ectomycorrhiza in plant N nutrition, proposed by Frank in 1885 (Martin and Botton, 1993), has received much attention recently. 3.2.1. Utilization of inorganic nitrogen by mycorrhizal fungi France and Reid (1983) indicated that ectomycorrhizal fungi can promote absorption of inorganic N by the roots of their host plants. Ammonium is a relatively good source of inorganic N for most ectomycorrhizal fungi, whereas nitrate is poorly assimilated by most taxa (Alexander, 1983; France and Reid, 1984; Plassard et al., 1991; Anderson et al., 1999). However, some ectomycorrhizal fungi, such as Paxillus involutus, Rhizopogon spp. and some isolates of Suillus placidus, grow well on nitrate and can be termed ‘nitrate fungi’ (Keller, 1996; Nygren et al., 2007). The mechanisms by which ectomycorrhizal fungi obtain inorganic N have been well studied. The enzymes involved in inorganic N assimilation include nitrate reductase, nitrite reductase, glutamine synthetase (GS), glutamate dehydrogenase (GDH) and glutamate synthase (GOGAT). Following nitrate reduction in the cells of the extramatrical mycelium and the fungal sheath, or ammonium uptake, the ammonium is incorporated into glutamate and glutamine by the action of GDH and GS, respectively. GOGAT may also play an important role in ammonium assimilation (Martin and Botton, 1993). 3.2.2. Utilization of organic nitrogen by ectomycorrhizal fungi Melin and Nilsson (1953) demonstrated that 15N labeled glutamate was absorbed by the mycelium of Boletus variegatus and that the nitrogen was transferred to the shoots of pine seedlings infected by the fungi in aseptic culture. Abuzinadah and Read (1988) compared the ability of three ectomycorrhizal fungi to obtain N from different amino acids as sole N sources. They found that aspartic and glutamic acids, arginine, alanine and serine all supported growth comparable to that of ammonium in each of the three fungi, whereas cystine, methionine, proline, threonine, tryptophane and tyrosine were not utilized. Ectomycorrhizal fungi appear to utilize the amide position N more easily than utilize the N which was bound in the amine of amino acids (Keller, 1996). Chalot and Brun (1998) hypothesized that amino acids can be directly absorbed by ectomycorrhizal fungi across the plasma membrane via amino acid-specific transport systems. Furthermore, small peptides consisting of 2e5 amino acids may also be transported across the plasma membrane via specific peptide transport systems. The fungal cell wall may contain cytosolic peptidase which

rapidly hydrolyzes these peptides into free amino acids. Amino acids can then be transferred to the host plants. Ectomycorrhizal fungi can assimilate proteins and transfer N derived from protein to their hosts (Abuzinadah and Read, 1986; Abuzinadah et al., 1986; Turnbull et al., 1995). Different fungi have different abilities to hydrolyze protein and transfer the products to plants. Abuzinadah and Read (1986) found that ectomycorrhizal fungi such as Suillus bovinus, Amanita muscaria, Paxillus involutus, Cenococcum geophilum, and Rhizopogon roseolus, which they called ‘protein fungi’, are able to use peptides and proteins as sole sources of N and so may be able to take up organic N and transfer it to their hosts. ‘Non-protein fungi’, however, such as Laccaria laccata and Lactarius rufus had little ability to grow on peptides and proteins but grew well on ammonium. Pisolithus tinctorius was placed in an intermediate category. More recent investigations have also shown that significant intraspecific variation exists with respect to the ability to utilize protein as an N source (Finlay et al., 1992; Keller, 1996; Nygren et al., 2007). Therefore, the classification of species to different categories may be misleading (Anderson et al., 1999). It was further demonstrated that different fungi have different abilities to transfer the N assimilated from protein to their host plants (Abuzinadah and Read, 1989a,b). Hebeloma crustuliniforme was more efficient at transferring assimilated N to their host plants than Paxillus involutus, which retained more of the N within its own tissues (Abuzinadah and Read, 1989a,b). The utilization of proteins by fungi requires the enzymatic degradation of proteins to peptides and amino acids before cellular uptake. Ectomycorrhizal fungi produce an acid carboxyl protease to hydrolyze protein into amino acids. A pH of about 3 is the optimum condition for this protease (Read, 1993). Proteases of ericoid (Leake and Read, 1990, 1991) and ectomycorrhizal (El-Badaoui and Botton, 1989; Zhu et al., 1994; Nygren et al., 2007) fungi have been detected and characterized. Zhu et al. (1994) also studied the factors influencing protease production by Hebeloma crustuliniforme. Substrate N concentration and pH are the most important factors influencing protease activity. Ectomycorrhizal fungi can metabolize amino acids actively and assimilate them via several N pathways (Chalot et al., 1994, 2006). Lindahl and Taylor (2004) studied the genetic potential of ectomycorrhizal fungi to produce N-acetylhexosaminidases, which hydrolyzes chitin to N-acetylglucosamine. Thus, N-acetylglucosamine and amino acids may replace ammonium and nitrate as the potential sources of nitrogen for ectomycorrhizal plants (Read and Perez-Moreno, 2003). Ericoid mycorrhizal fungi can utilize chitin as a sole source of N (Leake and Read, 1990; Mitchell et al., 1991; Kerley and Read, 1997), but most ectomycorrhizal fungi can only do so sparingly (Leake and Read, 1990). However, some ectomycorrhizal fungi can use glucosamine, which is the hydrolysis product of chitin. Lundeberg (1970) reported that seven isolates of saprotrophic fungi and 31 isolates of ectomycorrhizal fungi did not grow on glucosamine except for Tricholoma pessundatum and Boletus elegans. When ericoid mycorrhizal fungi grow together with ectomycorrhizal fungi, the chitinase produced by ericoid mycorrhizal fungi may allow ectomycorrhizal fungi to access the N in chitin (Mitchell et al., 1991). 3.2.3. Ectomycorrhizal fungi and polyphenol-protein complex Direct utilization of organic nitrogen by ectomycorrhizas, the ‘short-circuiting’ of the N cycle, may be important only when mineralization is not sufficient to meet plant uptake requirements (Northup et al., 1995b). However, mineralization and precipitation inputs appear to satisfy the great majority of the uptake requirements in temperate forest (see Table 4.2, in Alexander, 1983). Furthermore, most of the dissolved organic N in the forest floor is

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complexed by polyphenols (Qualls et al., 1991; Northup et al., 1995a) and may thus be largely unavailable to ectomycorrhizal fungi (Bending and Read, 1996b; Wu et al., 2003, 2005). Bending and Read (1996a) indicate that enzymatic breakdown of polyphenoleprotein complexes involves either peroxidases and polyphenol oxidases, or tannin carboxyl esterases. Saprotrophic fungi and ericoid mycorrhizal fungi have the ability to produce such enzymes, but most ectomycorrhizal fungi appear to have limited capacity relative to saprotrophs and ericoid mycorrhizal fungi (Bending and Read, 1997). It was thus proposed that utilization of such recalcitrant residues by ectomycorrhizal fungi is delayed until major changes are brought about by saprotrophic fungi (Bending and Read, 1996b). In a study of the use of proteinetannin complex as an N source, the pretreatment of the complex by saprotrophs did make its N available to ectomycorrhizal fungi (Wu et al., 2003). Therefore, saprotrophic microorganisms may play an important role for ectomycorrhizal fungi and their host plants by mobilizing N from recalcitrant organic N sources. 4. Interactions between saprotrophic microorganisms and ectomycorrhizal fungi When colonizing the same forest-floor substrates, saprotrophic microorganisms and ectomycorrhizal fungi may interact as competitors or facilitators (Fig. 2). Interactions between saprotrophic microorganisms and ectomycorrhizal fungi may influence growth and colonization of roots by ectomycorrhizal fungi (Shaw et al., 1995) and plant nutrition (Lindahl et al., 1999, 2002; Colparert and Van Laere, 1996; Colparert and Van Tichelen, 1996; Koide and Kabir, 2001). 4.1. Competition Saprotrophic fungi, such as some Trichoderma species and Collybia maculata, have been shown to inhibit ectomycorrhizal colonization (Summerbell, 1987; Shaw et al., 1995). The mechanisms for the inhibition are not clear. Summerbell (1987) suggested that Trichoderma can produce volatile and soluble antibiotics, which may inhibit a mycorrhizal fungus. Koide and Kabir (2001) indicated that at low N availability, forest-floor saprotrophic microorganisms significantly reduced the ability of the ectomycorrhizal fungus Pisolithus tinctorius to increase red pine N content. Therefore, it is possible for saprotrophic microorganisms to immobilize N even in the presence of ectomycorrhizal fungi. In contrast, ectomycorrhizal fungi may effectively compete against microorganisms for certain

1) Positive: Nutrition, MHB Ectomycorrhizal fungi 2) Negative: Trichoderma

3) Negative: Gadgil & Gadgil effects Saprotrophic microorganisms 4) Positive: Root exudates Fig. 2. Competition (dash line) and facilitation (solid line) between saprotrophic microorganisms and ectomycorrhizal fungi: 1) saprotrophs positively enhance ectomycorrhizal fungi through mobilization of nutrients, or heterotrophic mycorrhiza helper bacteria (MHB); 2) saprotrophs, such as Trichoderma species, inhibit ectomycorrhizal colonization; 3) ectomycorrhizal fungi retard litter decomposition through competing with saprotrophs for certain nutrients (Gadgil and Gadgil effects); 4) ectomycorrhizal fungi stimulate the activity of saprotrophs through the flow of carbon from ectomycorrhizal mycelia into the soil.

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nutrients (Gadgil and Gadgil, 1975) and thus retard litter decomposition (Gadgil and Gadgil, 1971). Koide and Wu (2003) suggested that ectomycorrhizas may compete with saprotrophic microorganisms for water and retard litter decomposition in dry conditions. Other research actually suggests that ectomycorrhizal fungi promote the activity of saprotrophic microorganisms (Entry et al., 1991, 1991; Dighton et al., 1987; Zhu and Ehrenfeld, 1996). 4.2. Facilitation The stimulation of other microorganisms by mycorrhizal fungi has been widely studied (Katznelson et al., 1962; Oswald and Ferchau, 1968; Griffiths et al., 1991; Setälä et al., 1999; Setälä, 2000). It was speculated, for example, that the flow of carbon through ectomycorrhizal mycelia into the soil (Linderman, 1988; Finlay and Söderstorm, 1992) can significantly stimulate other microorganisms (Katznelson et al., 1962; Oswald and Ferchau, 1968; Setälä et al., 1999; Setälä, 2000). Katznelson et al. (1962) indicated that the population of some ammonifying bacteria, actinomycetes and sugar-fermenting bacteria were greatly increased in the mycorrhizosphere. Setälä (2000) suggested that the stimulation of saprotrophic microorganisms would influence nutrient cycling and plant growth. Saprotrophic microorganisms may also benefit ectomycorrhizal fungi by improving ectomycorrhizal colonization, mobilizing and/ or mineralizing nutrients from recalcitrant organic matter and providing simple organic or mineral nutrients to mycorrhizal fungi and plants. Saprotrophic fungi are generally superior to ectomycorrhizal fungi in degrading lignin and other polyphenolics (Dighton, 1991). Therefore, ectomycorrhizal fungi may gain access to nutrients in recalcitrant forms of litter if they are in close association with saprotrophs (Colparert and Van Laere, 1996; Lindahl et al., 2001). Some heterotrophic microorganisms, such as mycorrhiza helper bacteria (MHB), can also promote ectomycorrhizal colonization (Garbaye and Bowen, 1989; Garbaye, 1994). 5. Concluding thoughts The ability of ectomycorrhizal fungi to obtain N from organic forms such as amino acids and proteins under controlled laboratory systems is well established (Melin and Nilsson, 1953; Abuzinadah and Read, 1986; Finlay et al., 1992; Turnbull et al., 1995; Bending and Read, 1996b; Wu et al., 2005). However, the importance of ectomycorrhizal fungi to obtain N from organic forms in temperate forest ecosystems remains questionable. Most of the N in many temperate forests is dominated by polyphenoleprotein complexes, which are difficult to use by ectomycorrhizal fungi (Bending and Read, 1997; Colparert and Van Laere, 1996; Colparert and Van Tichelen, 1996; Wu et al., 2005). Compared to ectomycorrhizal fungi, saprotrophic microorganisms possess a much greater capacity to mobilize N from the polyphenoliceorganic N complexes such as the tannineprotein complex, thus may potentially influence the N nutrition of ectomycorrhizal fungi and their associated plants (Bending and Read, 1997; Wu et al., 2005; Wurzburger and Hendrick, 2009). Our knowledge on how saprotrophic microorganisms influence ectomycorrhizal fungi in N nutrition is currently limited (Baar and Stanton, 2000; Cairney and Meharg, 2002; Lindahl et al., 2007). The interactions between saprotrophic microorganisms and ectomycorrhizal fungi may play important roles in the N economy in temperate forest ecosystems. First, the immobilization/mineralization of N by saprotrophs is controlled by C:N ratio. Plants and other phototrophic organisms provide initial C sources in ecosystems. It has been reported that roots release a wide range of carbon compounds of low molecular weight amounting to between 10%

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and 20% of total net fixed carbon (Rovira, 1991). Thus, plants strongly influence the soil C:N ratio and the potential rates of net N mineralization and N immobilization (Knops et al., 2002; Lovett et al., 2004; Chapman et al., 2005). Limitations on plant C assimilation may indirectly impose limitations on soil microorganisms (Högberg et al., 2003), thus may regulate saprotrophs mineralizing N in order to trade C requirements for the growth of saprotrophs (Fig. 3). Second, certain saprotrophs decrease the C:N ratio in the litter of upper forest-floor layers through respiration removing C and immobilization retaining N (Fig. 3). When C:N ratio decrease to a critical level for N mineralization, saprotrophs release inorganic N for plant growths. Whereas, ectomycorrhizal fungi mobilize N in the fragmented litter and humus (Lindahl et al., 2007). The spatial separation of certain saprotrophic and ectomycorrhizal fungi in boreal forest soils allows degradation of litter components and mobilization of nutrients occurring at different boreal forest-floor layers, thus maintain the partitioning of nutrients by the fungal communities (Lindahl et al., 2007). Partitioning resources through the interactions between saprotrophic microorganisms and ectomycorrhizal fungi may allow each component to utilize N resources economically especially in the N-limited temperate forest ecosystems. On the other hand, ectomycorrhizal fungi may also live together with some other saprotrophs. Under this circumstance, ectomycorrhizal fungi may access some mobilized N by saprotrophs from polyphenoleprotein complexes (Colparert and Van Laere,

1996; Lindahl et al., 2001), therefore, compete with these saprotrophs for organic N. Third, plants obtain C from photosynthesis and do not necessarily rely on C included in the organic N in the forest floor as their C sources. Furthermore, plants are reported allocating C to mycorrhizal fungi when limited by nutrients such as N or P (Finlay and Söderstorm, 1992; Treseder and Allen, 2002; Hobbie, 2006). In Nlimited temperate forests, therefore, the symbiotic ectomycorrhizal fungi obtain C from their host plants. In contrast, saprotrophs obtain C through decomposing all possible forms of organic matter in the forest floor and provide mineralized N to plants. If both inorganic N and organic N are available, plants and associated ectomycorrhizal fungi may economically use small molecules of inorganic N mineralized by saprotrophs instead of spending more energy to compete with saprotrophs for large molecules of organic N as well as the unlimited C contained within those large molecules. Harrison et al. (2007) reported that all of the plants tested in a temperate grassland preferred inorganic over organic N is most likely a reflection of the relatively high rate of microbial N mineralization. Similar situation may exist in the temperate forest ecosystems. Finally, in natural ecosystems interactions among organisms are dynamic. For example, ectomycorrhizal fungi form facultative mutualistic associations with plants, thus may convert from a mutulistic to parasitic association (Johnson et al., 1997; Lambers et al., 2009) with respect to their host benefits. Similarly,

Fig. 3. A diagram of C and N economy for plants, ectomycorrhizal fungi and saprotrophs. 1) photosynthesis providing organic C from CO2 to plants; 2) respiration pathway returning organic C into CO2; 3) under high C:N ratio, saprothophs immobilize N and release CO2 from organic C (litter) through respiration; 4) under low C:N ratio, saprothophs mineralize N providing N to plants and may obtain C from plants; 5) ectomycorrhizal fungi obtain organic C from plants, and provide N in inorganic forms (NIno) and small molecules of organic N (NOrg),and phosphorus (P) to their host plants; 6) plants directly obtain inorganic N from soil; 7) abilities of plants and ectomycorrhizal fungi in obtaining N directly from organic N complexes (polyphenoleprotein complexes) in temperate forests are not fully understood. Interactions between saprotrophic microorganisms (Sap) and ectomycorrhizal fungi (ECM) may be important in the organic N nutrition.

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ectomycorrhizal fungi may also shift from biotrophy to saprotrophy (Boucher et al., 1982; Dighton, 2007; Koide et al., 2008; Read and Perez-Moreno, 2003) depending on their capacity to obtain C. It is reported that when ectomycorrhizal fungi cannot benefit their hosts, plants may limit C, requiring that some ectomycorrhizal fungi metabolize C from litter as saprotrophs (Talbot et al., 2008). Under these circumstances, previously biotrophic ectomycorrhizal fungi may saprotrophically explore C resources from polyphenoleorganic N complexes in the forest floor, therefore may mobilize N from the polyphenoliceorganic N complexes and further mineralize N into inorganic forms for the N nutrition of plants. Although more and more evidence suggests that ectomycorrhizal fungi can obtain N from organic forms under certain experimental conditions, circumventing the N mineralization pathway by ectomycorrhizal fungi and the role of saprotrophic microorganisms in this process require further investigation. Understanding interactions between saprotrophic microorganisms and ectomycorrhizal fungi is a priority for clarifying N cycling processes in temperate forest ecosystems. Recently developed stable isotope probing (SIP) may be useful in understanding microbial-mediated flows of carbon and nitrogen in the environment (Neufeld et al., 2007), such as phenol biodegradation (DeRito and Madsen, 2009) and protein catabolism (Jehmlich et al., 2008). The application of DNA-based stable isotope probing (DNA-SIP) by assessment of isotopes such as 15N and 13C accumulating in the DNA of saprotrophic microorganisms and ectomycorrhizal fungi may provide evidence of the relevant N transformation. Acknowledgements The author would like to thank the A. W. Mellon Foundation and the Department of Horticulture, Pennsylvania State University, for financial support. The author would also like to thank Dr. Roger T. Koide for his critical comments and revisions, and three anonymous for their editorial comments on this manuscript. References Aber, J.D., Nadelhoffer, K.J., Steudler, P., Melillo, J.M., 1989. Nitrogen saturation in northern forest ecosystems. BioScience 39, 378e386. Aber, J.D., 1992. Nitrogen cycling and nitrogen saturation in temperate forest ecosystem. Trends in Ecology and Evolution 7, 220e223. Abuzinadah, R.A., Read, D.J., 1986. The role of proteins in the nitrogen nutrition of ectomycorrhizal plants. I. Utilization of peptides and proteins by ectomycorrhizal fungi. New Phytologist 103, 481e493. Abuzinadah, R.A., Finlay, R.D., Read, D.J., 1986. The role of proteins in the nitrogen nutrition of ectomycorrhizal plants. II. Utilization of peptides and proteins by ectomycorrhizal plants of Pinus contorta. New Phytologist 103, 495e506. Abuzinadah, R.A., Read, D.J., 1988. Amino acids as nitrogen sources for ectomycorrhizal fungi: utilization of individual amino acids. Transactions of the British Mycological Society 91, 473e479. Abuzinadah, R.A., Read, D.J., 1989a. The role of proteins in the nitrogen nutrition of ectomycorrhizal plants. IV. The utilization of peptides by birch (Betula pendula L.) infected with different mycorrhizal fungi. New Phytologist 112, 55e60. Abuzinadah, R.A., Read, D.J., 1989b. The role of proteins in the nitrogen nutrition of ectomycorrhizal plants. V. Nitrogen transfer in birch (Betula pendula) grown in association with mycorrhizal and non-mycorrhizal fungi. New Phytologist 112, 61e68. Abuarghub, S.M., Read, D.J., 1988. The biology of mycorrhiza in the Ericaceae. XI. The distribution of nitrogen in soil of a typical upland Callunetum with special reference to the ‘free’ amino acids. New Phytologist 108, 425e431. Alexander, I.J., 1983. The significance of ectomycorrhizas in the nitrogen cycle. In: Lea, J.A., McNeill, S., Rovison, I.H. (Eds.), Nitrogen as an Ecological Factor. Blackwell, Oxford, UK, pp. 69e93. Allen, E.B., Allen, M.F., Helm, D.J., Trappe, J.M., Molina, R., Rincon, E., 1995. Patterns and regulation of mycorrhizal plant and fungal diversity. Plant and Soil 170, 47e62. Anderson, I.C., Chambers, S.M., Cairney, J.W.G., 1999. Intra- and interspecific variation in pattern of organic and inorganic nitrogen utilization by three Australian Pisolithus species. Mycological Research 103, 1579e1587. Appel, T., Mengel, K., 1990. Importance of organic nitrogen fractions in sandy soils, obtained by electro-ultrafiltration or CaCl2-extraction for nitrogen mineralization and nitrogen uptake of rape. Biology and Fertility of Soil 10, 97e101.

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