Conservation of Edible Ectomycorrhizal Mushrooms: Understanding of the ECM Fungi Mediated Carbon and Nitrogen Movement within Forest Ecosystems

*Lu-Min Vaario and Norihisa Matsushita*

### **Abstract**

Most edible ectomycorrhizal (ECM) mushrooms are currently harvested from nature and many of them are high-priced. Demand for the wild mushrooms as a culinary delicacy has stimulated research that aims to understand (1) the puzzled role that the ECM fungi play in the forest ecosystem, and (2) nutritional and other requirements for fruiting, which is highly variable. In this review, we focus on understanding of the ECM fungi mediated carbon and nitrogen movement between the symbiotic partners and on the interactions with other fungi in forest ecosystems. Thereby, we better understand the diverse nitrogen requirements for edible ECM fungal growth and mushroom fruiting. We attempt to provide a theoretical basis for the future research of edible ECM mushrooms in wild and controlled conditions.

**Keywords:** culture, cultivation, ectomycorrhizal fungi, edible mushrooms, nitrogen uptake

#### **1. Introduction**

Forests play a crucial role in the global environment and economy. Forest-based wood products as well as non-wood forest products have offered remarkable resources and benefits for the well-being of people [1, 2]. A healthy and wellgrowing forest system is largely dependent on available soil nutrients and efficient nutrient cycling [3, 4], especially nitrogen (N). As we know, nitrogen is a limiting resource for plant growth in many temperate forests.

Nitrogen is necessary for plants. Most crops require N relatively high amounts, but only a small amount of available N is present in soil at a time. A large source of soil N is the atmospheric dinitrogen (N2), the major gas of air (79%) [5]. Only certain microorganisms can bind molecular nitrogen from air. All other organisms need to take up nitrogen from soil. Soil organic matter (especially humus) acts as a storage and supplier of nitrogen for plant roots and microorganisms;

almost 90–95% of soil total nitrogen originates from soil organic matter [6, 7]. Plants acquire N mostly from the inorganic forms such as ammonium and nitrate. However, plants that associate with mycorrhizal fungi are considered to have greater access to organic nitrogen pools when compared to non-mycorrhizal plants [5].

ECM fungi play an important role in the nutrient cycle of terrestrial ecosystems. Especially in a forest poor in nutrients, the growth of trees depends on the existence of mycorrhizal fungi. The value of ECM fungi is evaluated from the global framework. ECM fungi provide hidden biological fertilizers for increasing plant biomass, conventional afforestation, and ecosystem restoration practices; they also control soil pathogens [8–10].

In addition to benefits for forests, many ECM fungi produce edible mushrooms that are widely appreciated for their nutritional, medicinal, and gastronomic properties [11]. One of the major challenges of the twenty-first century is to produce sufficient food. From that perspective, wild mushrooms as non-wood forest products are getting more and more attention globally [12]. It would be convenient if these mushrooms could be cultivated. However, most edible ECM mushrooms can only be collected from nature and not cultivated artificially [11]. The main obstacle to the cultivation of edible ECM mushroom is their need to be associated with a host plant in plantations. The association is obligatory for the successful growth and fruiting of the mushrooms. The unanimous discussion of the nutritional growth requirements of ECM edible fungi is a topic of interest for scientists.

An in-depth understanding of the nutritional requirements of ECM fungi and the role of ECM fungi in nutrient cycling, particularly in ECM fungi mediating carbon and nitrogen movement within forest ecosystems will be summarized in this chapter. The nutritional requirements to successfully culture and cultivate ECM fungi will be discussed.

#### **2. Ectomycorrhizal fungi**

#### **2.1 Ectomycorrhizal fungi**

Ectomycorrhizal fungi are found in association with the roots of most forest trees throughout the world. ECM fungi form obligate symbioses with many of the dominant trees in temperate and boreal forest, as well as in some tropical forests. ECM fungi do not penetrate their host's cell walls. Instead, they form an entirely intercellular interface, known as Hartig net, consisting of highly branched hyphae that forms a latticework between epidermal and cortical cells [13]. Hartig net provides a large surface area for the two symbiotic partners and it is the site of nutrient exchange. Carbon (C) is transported to the fungus from a tree that receives limiting nutrients in exchange. The fungus can transport nutrients beyond the nutrient depletion zone surrounding the host's root system and release from immobilized sources inaccessible to the plant [13, 14]. ECM fungi are thus regarded as key elements of forest nutrient cycles and as strong drivers of forest ecosystem processes [15].

Most (86%) terrestrial plant species obtain mineral nutrients through mycorrhizal symbionts as estimated using taxonomic and ecological extrapolation methods [16]. An estimate of ECM fungal species richness is likely between 20,000 and 25,000 [16, 17]. These ECM fungi belong to more than 80 independently evolved lineages and to more than 250 genera, mainly in Basidiomycetes and Ascomycetes [18].

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**ecosystems**

*Conservation of Edible Ectomycorrhizal Mushrooms: Understanding of the ECM Fungi…*

matter decomposition, nutrient cycling, and carbon sequestration [19–21].

Ectomycorrhizal fungi are essential contributors in forest ecosystems by forming beneficial symbiosis plants. These fungi drive forest soil processes such as soil organic

ECM fungi have the ability to provide hosts not only nitrogen but a variety of other major nutrients, including phosphorus, potassium, calcium, magnesium, sulfur, as well as micronutrients such as iron, zinc, copper, and manganese. However, they are often ignored because N is the main growth-limiting element in many forest ecosystems, particularly in the Northern Hemisphere [19, 22, 23]. In addition to nutrients, trees receive several other benefits. First, the resistance of trees against pathogens is improved due to the mycelial network [24]. Second, the ECM mycelial networks are involved in water transport [25]. Third, ECM fungi can relieve salt and heavy metal stress of the host plants [9]. The benefits that the ECM fungi offer are complicatedly regulated by the host type, ECM species, as well as climatic and environmental conditions. Recently, a study based on a climate change model predicted that the global abundance of ECM-associated trees will decline by 10% by the end of 2070, and the majority of this will take place in boreal and temperate ecotones [26]. Therefore, the conservation of ECM fungi should be taken

Fungal mycelium has been estimated as one of the largest living organisms on Earth [27]. Hyphae is composed of fungal mycelium and other structures including rhizomorphs. Rhizomorphs are structures through which fungi can spread in their environment and search for new substrates to colonize. The structure of ectomycorrhiza is diverse. Agerer [28] proposed that ECM mycelia systems influence on their patterns of differentiation and putative ecological importance. Mycorrhizal fungi have been classified into four exploitation types depending on the extent of hyphal development: contact, short-distance, medium-distance, and long-distance.

ECM fungi are characterized according to the water repellence of the mycelium. Fungi vary from extremely hydrophobic to extremely hydrophilic types [29]. All fungal growth parameters such as hyphal hydrophilicity, presence of rhizomorphs, and mat formation correspond together to how fungi interact with and exploit the environment [28, 30]. The function of extraradical mycelia of ECM fungi is the transportation of nutrients between plant and soil environment [13, 31].

Ectomycorrhizas differ in their ability to take up and transport nutrients, and thus, promote tree growth [32, 33]. The differences in ECM effectiveness are often species specific or even strain specific [34]. It is evident that the amount and differentiation of extraradical mycelium is an important ecological factor for tree

**3. Contribution of ectomycorrhizal fungi to nitrogen cycling in forest** 

sources. Atmospheric nitrogen deposition is an external source, and the living organisms and their decomposition products are an internal source [39, 40].

The major N sources in the forest floor can be divided into external and internal

**2.2 General roles of ectomycorrhizal fungi in forest ecosystems**

*DOI: http://dx.doi.org/10.5772/intechopen.95399*

as an important issue.

**2.3 Structure of ectomycorrhizas is diverse**

performance [35–37] and soil nutrition [38].

**3.1 Forms of nitrogen in forest soil**

*Conservation of Edible Ectomycorrhizal Mushrooms: Understanding of the ECM Fungi… DOI: http://dx.doi.org/10.5772/intechopen.95399*

#### **2.2 General roles of ectomycorrhizal fungi in forest ecosystems**

Ectomycorrhizal fungi are essential contributors in forest ecosystems by forming beneficial symbiosis plants. These fungi drive forest soil processes such as soil organic matter decomposition, nutrient cycling, and carbon sequestration [19–21].

ECM fungi have the ability to provide hosts not only nitrogen but a variety of other major nutrients, including phosphorus, potassium, calcium, magnesium, sulfur, as well as micronutrients such as iron, zinc, copper, and manganese. However, they are often ignored because N is the main growth-limiting element in many forest ecosystems, particularly in the Northern Hemisphere [19, 22, 23]. In addition to nutrients, trees receive several other benefits. First, the resistance of trees against pathogens is improved due to the mycelial network [24]. Second, the ECM mycelial networks are involved in water transport [25]. Third, ECM fungi can relieve salt and heavy metal stress of the host plants [9]. The benefits that the ECM fungi offer are complicatedly regulated by the host type, ECM species, as well as climatic and environmental conditions. Recently, a study based on a climate change model predicted that the global abundance of ECM-associated trees will decline by 10% by the end of 2070, and the majority of this will take place in boreal and temperate ecotones [26]. Therefore, the conservation of ECM fungi should be taken as an important issue.

#### **2.3 Structure of ectomycorrhizas is diverse**

Fungal mycelium has been estimated as one of the largest living organisms on Earth [27]. Hyphae is composed of fungal mycelium and other structures including rhizomorphs. Rhizomorphs are structures through which fungi can spread in their environment and search for new substrates to colonize. The structure of ectomycorrhiza is diverse. Agerer [28] proposed that ECM mycelia systems influence on their patterns of differentiation and putative ecological importance. Mycorrhizal fungi have been classified into four exploitation types depending on the extent of hyphal development: contact, short-distance, medium-distance, and long-distance.

ECM fungi are characterized according to the water repellence of the mycelium. Fungi vary from extremely hydrophobic to extremely hydrophilic types [29]. All fungal growth parameters such as hyphal hydrophilicity, presence of rhizomorphs, and mat formation correspond together to how fungi interact with and exploit the environment [28, 30]. The function of extraradical mycelia of ECM fungi is the transportation of nutrients between plant and soil environment [13, 31].

Ectomycorrhizas differ in their ability to take up and transport nutrients, and thus, promote tree growth [32, 33]. The differences in ECM effectiveness are often species specific or even strain specific [34]. It is evident that the amount and differentiation of extraradical mycelium is an important ecological factor for tree performance [35–37] and soil nutrition [38].

#### **3. Contribution of ectomycorrhizal fungi to nitrogen cycling in forest ecosystems**

#### **3.1 Forms of nitrogen in forest soil**

The major N sources in the forest floor can be divided into external and internal sources. Atmospheric nitrogen deposition is an external source, and the living organisms and their decomposition products are an internal source [39, 40].

*Nitrogen in Agriculture - Physiological, Agricultural and Ecological Aspects*

ments of ECM edible fungi is a topic of interest for scientists.

plants [5].

soil pathogens [8–10].

fungi will be discussed.

**2. Ectomycorrhizal fungi**

**2.1 Ectomycorrhizal fungi**

almost 90–95% of soil total nitrogen originates from soil organic matter [6, 7]. Plants acquire N mostly from the inorganic forms such as ammonium and nitrate. However, plants that associate with mycorrhizal fungi are considered to have greater access to organic nitrogen pools when compared to non-mycorrhizal

ECM fungi play an important role in the nutrient cycle of terrestrial ecosystems. Especially in a forest poor in nutrients, the growth of trees depends on the existence of mycorrhizal fungi. The value of ECM fungi is evaluated from the global framework. ECM fungi provide hidden biological fertilizers for increasing plant biomass, conventional afforestation, and ecosystem restoration practices; they also control

In addition to benefits for forests, many ECM fungi produce edible mushrooms that are widely appreciated for their nutritional, medicinal, and gastronomic properties [11]. One of the major challenges of the twenty-first century is to produce sufficient food. From that perspective, wild mushrooms as non-wood forest products are getting more and more attention globally [12]. It would be convenient if these mushrooms could be cultivated. However, most edible ECM mushrooms can only be collected from nature and not cultivated artificially [11]. The main obstacle to the cultivation of edible ECM mushroom is their need to be associated with a host plant in plantations. The association is obligatory for the successful growth and fruiting of the mushrooms. The unanimous discussion of the nutritional growth require-

An in-depth understanding of the nutritional requirements of ECM fungi and the role of ECM fungi in nutrient cycling, particularly in ECM fungi mediating carbon and nitrogen movement within forest ecosystems will be summarized in this chapter. The nutritional requirements to successfully culture and cultivate ECM

Ectomycorrhizal fungi are found in association with the roots of most forest trees throughout the world. ECM fungi form obligate symbioses with many of the dominant trees in temperate and boreal forest, as well as in some tropical forests. ECM fungi do not penetrate their host's cell walls. Instead, they form an entirely intercellular interface, known as Hartig net, consisting of highly branched hyphae that forms a latticework between epidermal and cortical cells [13]. Hartig net provides a large surface area for the two symbiotic partners and it is the site of nutrient exchange. Carbon (C) is transported to the fungus from a tree that receives limiting nutrients in exchange. The fungus can transport nutrients beyond the nutrient depletion zone surrounding the host's root system and release from immobilized sources inaccessible to the plant [13, 14]. ECM fungi are thus regarded as key elements of forest nutrient cycles and as strong drivers of forest ecosystem

Most (86%) terrestrial plant species obtain mineral nutrients through mycor-

rhizal symbionts as estimated using taxonomic and ecological extrapolation methods [16]. An estimate of ECM fungal species richness is likely between 20,000 and 25,000 [16, 17]. These ECM fungi belong to more than 80 independently evolved lineages and to more than 250 genera, mainly in Basidiomycetes and

**152**

processes [15].

Ascomycetes [18].

Ammonium and nitrate are the two major pools of inorganic N. Ammonium is most often the dominant inorganic N pool available to trees in coniferous ecosystems. Nitrate concentrations are usually relatively low in mature forests [41].

Most of the nitrogen in forest soils is bound to organic compounds [42]. It is well known that over 90% of N occurs in organic forms in most surface soils [7, 43]. The forms of organic N can be roughly divided into two categories. (I) Organic residues consisting of undecomposed animal and plant residues and partial decomposition products, and (ii) soil organic matter or humus. The humus is composed of nonhumic, easily identifiable compounds (e.g. amino acids, carbohydrates, nucleic acids, etc.) and complex humic substances, such as high-molecular-weight amorphous and aromatic compounds, formed during the decomposition process. The importance of humus is widely recognized in maintaining and improving soil fertility [7].

The distribution of major N compounds was investigated in different climatic and geological conditions including arctic, cool, temperate, subtropical, and tropical climates early [44]. The results indicated that about 33–42% of soil N occurs as free and protein amino acids. The amino acid composition of all soils, however, was remarkably similar. The composition and concentration of amino acids has shown generally constant throughout the growing season [45], which suggests that amino acids originate from a common source or through similar biochemical processes. However, the distribution of N compounds at different regions seems to be related to decomposition process and as well as forest types [46]. Soil proteins are often not free, they are bound to humic compounds and are not soluble. These N forms cannot directly be used by plants, they need to be depolymerized by microorganisms and converted into plant available monomeric organic or mineral N forms.

#### **3.2 Diversity in nitrogen uptake in Ectomycorrhizal Fungi**

Ectomycorrhizas occur widely in forest ecosystems. Most of the terrestrial plant species are in symbiosis with mycorrhizal fungi, about 3% of them are ectomycorrhizal. The most common tree species belong to Pinaceae, Salicaceae, Betulaceae, Fagaceae and Myrtaceae [13, 47]. The general mechanism of ECM fungi to improve plant nutrition is the so called Hartig net structure that increase the surface area of roots to absorb nutrients.

Ectomycorrhizal fungi are able to take up both inorganic and organic forms of N. Ammonium is generally recognized as the most readily utilizable form for most ECM fungi when studied in mycelial cultures [48, 49] or with ECM roots in vitro and in the field [50]. Nygren and colleagues [51] demonstrated that 68 species of ECM fungi used nitrate as the sole N source in a pure culture. However, the pure culture conditions do not reflect the N preference of ECM fungi in nature [52]. *Laccaria laccata* was shown to uptake nitrate and transfer it to the host plant when in nitrate-rich conditions [53].

In other studies, ECM seedlings demonstrate a strong preference for amino acids over ammonium [54]. Already in 1953, Melin and Nilsson [55] demonstrated that 15N labelled glutamate was absorbed by the mycelium of *Boletus variegatus*, and that the nitrogen was transferred to the shoots of pine seedlings that had been infected with the fungi in an aseptic culture. Many ECM fungi are able to grow with amino acids as the N source in pure culture and also in association with host trees [56–59].

The capacities of ECM fungi to mineralize organic N differ. Abuzinadah and Read [60] found that ECM fungi such as *Suillus bovinus*, *Amanita muscaria*, *Paxillus involutus*, *Cenococcum geophilum*, and *Rhizopogon roseolus* were able to use peptides and proteins as their sole N sources. In contrast, *Laccaria laccata* and *Lactarius rufus* had little ability to grow with peptides and proteins but they grew well with

**155**

*Conservation of Edible Ectomycorrhizal Mushrooms: Understanding of the ECM Fungi…*

ammonium. It was further demonstrated that different fungal species, even different strains had different abilities to utilize organic N and/or transfer the assimilated N to their host plants [60]. Some ECM fungi might take up the nitrogen compound completely and some break the molecules into smaller organic or inorganic forms. The difference in the ability of ECM fungi to transfer N from chitin, protein, and other organic substances in litter and humus was explained by differences in their

ECM fungi have several functionally distinct metabolic pathways to transfer N. ECM fungal hyphal morphology, species niche (original living conditions), genetic characteristics and carbon costs to host plants may influence on their capacity to

ECM fungal hyphae morphology is diverse. Morphology seems to have a great influence on the hyphal enzymatic ability of ECM fungi. ECM species with hydrophilic ectomycorrhizal hyphae have proteolytic activities and they are adapted to N-limited conditions [62]. In contrast, other ECM fungi with hydrophobic ectomycorrhizal hyphae, similar to many saprotrophic fungi, form aggregated hyphae (rhizomorphs) for long-distance transport of elements. This is presumably an

In addition to hydrophobicity, another aspect is to consider the size of mycelia.

The species that form extensive extraradical mycelia (e.g. *Cortinarius*, *Suillus*, *Tricholoma* species) have different capacity to utilize organic N than those species that form diffuse, spatially limited extraradical mycelia (e.g. *Amanita*, *Lactarius* species). These differences in mycelia are thought to be associated with different reproductive and colonization strategies [58, 62]. It is believed that extensive mycelia are established infrequently, but it is long- living. In contrary, the diffuse mycelia become more stable, usually by spores for the generation, but the mycelia do not persist. The long-living extraradical mycelia is believed to be more efficient

Studies based on the stable N isotope ratios in ECM fungal fruitbodies have provided new insights and evidence for the N sources of ECM fungi. As we know, the relative abundance of stable isotopes in food webs follows from discrimination against heavier isotopes in several biochemical processes [64]. The ratio is useful particularly in studying nitrogen cycling mediated by mycorrhizal fungi [65]. Stable N isotope ratios in ECM fungal fruitbodies showed that those having long-living

Thus, the signature of 15N in ECM fruitbodies was determined by the morphological characteristics of the mycelia. Another observation revealed by the isotope studies is that ECM fungal species that can utilize organic N exhibited higher δ15N in their fruitbodies than those that are restricted to mineral N sources [67, 68].

The form of nitrogen in the environment influences N mobilization by ECM fungi. The species common in low inorganic N soils grew well with protein, glutamine, and serine whereas species in high inorganic N soils grew well with glutamine, but poorly with protein and serine [67]. Differences among ECM fungal species in their ability to access and take up different N forms indicate that the form and abundance of N in the environment may be a defining factor for ECM fungal species niche [69]. ECM species are selected by the N form that is predominant in

*3.2.2 Nitrogen utilization of ECM fungi is related to the nitrogen status* 

15N than those having short-living mycelia [58, 66, 67].

*3.2.1 Mycelium structure determines the efficiency of ECM transport nitrogen*

*DOI: http://dx.doi.org/10.5772/intechopen.95399*

enzyme secretion profiles [61].

utilize and mineralize organic N.

adaptation for patchily distributed resources [63].

to process N than short-living mycelia.

mycelia exhibited higher δ

*of the habitat*

#### *Conservation of Edible Ectomycorrhizal Mushrooms: Understanding of the ECM Fungi… DOI: http://dx.doi.org/10.5772/intechopen.95399*

ammonium. It was further demonstrated that different fungal species, even different strains had different abilities to utilize organic N and/or transfer the assimilated N to their host plants [60]. Some ECM fungi might take up the nitrogen compound completely and some break the molecules into smaller organic or inorganic forms. The difference in the ability of ECM fungi to transfer N from chitin, protein, and other organic substances in litter and humus was explained by differences in their enzyme secretion profiles [61].

ECM fungi have several functionally distinct metabolic pathways to transfer N. ECM fungal hyphal morphology, species niche (original living conditions), genetic characteristics and carbon costs to host plants may influence on their capacity to utilize and mineralize organic N.

#### *3.2.1 Mycelium structure determines the efficiency of ECM transport nitrogen*

ECM fungal hyphae morphology is diverse. Morphology seems to have a great influence on the hyphal enzymatic ability of ECM fungi. ECM species with hydrophilic ectomycorrhizal hyphae have proteolytic activities and they are adapted to N-limited conditions [62]. In contrast, other ECM fungi with hydrophobic ectomycorrhizal hyphae, similar to many saprotrophic fungi, form aggregated hyphae (rhizomorphs) for long-distance transport of elements. This is presumably an adaptation for patchily distributed resources [63].

In addition to hydrophobicity, another aspect is to consider the size of mycelia. The species that form extensive extraradical mycelia (e.g. *Cortinarius*, *Suillus*, *Tricholoma* species) have different capacity to utilize organic N than those species that form diffuse, spatially limited extraradical mycelia (e.g. *Amanita*, *Lactarius* species). These differences in mycelia are thought to be associated with different reproductive and colonization strategies [58, 62]. It is believed that extensive mycelia are established infrequently, but it is long- living. In contrary, the diffuse mycelia become more stable, usually by spores for the generation, but the mycelia do not persist. The long-living extraradical mycelia is believed to be more efficient to process N than short-living mycelia.

Studies based on the stable N isotope ratios in ECM fungal fruitbodies have provided new insights and evidence for the N sources of ECM fungi. As we know, the relative abundance of stable isotopes in food webs follows from discrimination against heavier isotopes in several biochemical processes [64]. The ratio is useful particularly in studying nitrogen cycling mediated by mycorrhizal fungi [65]. Stable N isotope ratios in ECM fungal fruitbodies showed that those having long-living mycelia exhibited higher δ 15N than those having short-living mycelia [58, 66, 67].

Thus, the signature of 15N in ECM fruitbodies was determined by the morphological characteristics of the mycelia. Another observation revealed by the isotope studies is that ECM fungal species that can utilize organic N exhibited higher δ15N in their fruitbodies than those that are restricted to mineral N sources [67, 68].

#### *3.2.2 Nitrogen utilization of ECM fungi is related to the nitrogen status of the habitat*

The form of nitrogen in the environment influences N mobilization by ECM fungi. The species common in low inorganic N soils grew well with protein, glutamine, and serine whereas species in high inorganic N soils grew well with glutamine, but poorly with protein and serine [67]. Differences among ECM fungal species in their ability to access and take up different N forms indicate that the form and abundance of N in the environment may be a defining factor for ECM fungal species niche [69]. ECM species are selected by the N form that is predominant in

*Nitrogen in Agriculture - Physiological, Agricultural and Ecological Aspects*

Nitrate concentrations are usually relatively low in mature forests [41].

humus is widely recognized in maintaining and improving soil fertility [7].

**3.2 Diversity in nitrogen uptake in Ectomycorrhizal Fungi**

Ammonium and nitrate are the two major pools of inorganic N. Ammonium is most often the dominant inorganic N pool available to trees in coniferous ecosystems.

Most of the nitrogen in forest soils is bound to organic compounds [42]. It is well known that over 90% of N occurs in organic forms in most surface soils [7, 43]. The forms of organic N can be roughly divided into two categories. (I) Organic residues consisting of undecomposed animal and plant residues and partial decomposition products, and (ii) soil organic matter or humus. The humus is composed of nonhumic, easily identifiable compounds (e.g. amino acids, carbohydrates, nucleic acids, etc.) and complex humic substances, such as high-molecular-weight amorphous and aromatic compounds, formed during the decomposition process. The importance of

The distribution of major N compounds was investigated in different climatic and geological conditions including arctic, cool, temperate, subtropical, and tropical climates early [44]. The results indicated that about 33–42% of soil N occurs as free and protein amino acids. The amino acid composition of all soils, however, was remarkably similar. The composition and concentration of amino acids has shown generally constant throughout the growing season [45], which suggests that amino acids originate from a common source or through similar biochemical processes. However, the distribution of N compounds at different regions seems to be related to decomposition process and as well as forest types [46]. Soil proteins are often not free, they are bound to humic compounds and are not soluble. These N forms cannot directly be used by plants, they need to be depolymerized by microorganisms and converted into plant available monomeric organic or mineral

Ectomycorrhizas occur widely in forest ecosystems. Most of the terrestrial plant species are in symbiosis with mycorrhizal fungi, about 3% of them are ectomycorrhizal. The most common tree species belong to Pinaceae, Salicaceae, Betulaceae, Fagaceae and Myrtaceae [13, 47]. The general mechanism of ECM fungi to improve plant nutrition is the so called Hartig net structure that increase the surface area of

Ectomycorrhizal fungi are able to take up both inorganic and organic forms of N. Ammonium is generally recognized as the most readily utilizable form for most ECM fungi when studied in mycelial cultures [48, 49] or with ECM roots in vitro and in the field [50]. Nygren and colleagues [51] demonstrated that 68 species of ECM fungi used nitrate as the sole N source in a pure culture. However, the pure culture conditions do not reflect the N preference of ECM fungi in nature [52]. *Laccaria laccata* was shown to uptake nitrate and transfer it to the host plant when

In other studies, ECM seedlings demonstrate a strong preference for amino acids over ammonium [54]. Already in 1953, Melin and Nilsson [55] demonstrated that 15N labelled glutamate was absorbed by the mycelium of *Boletus variegatus*, and that the nitrogen was transferred to the shoots of pine seedlings that had been infected with the fungi in an aseptic culture. Many ECM fungi are able to grow with amino acids as the N source in pure culture and also in association with host trees [56–59]. The capacities of ECM fungi to mineralize organic N differ. Abuzinadah and Read [60] found that ECM fungi such as *Suillus bovinus*, *Amanita muscaria*, *Paxillus involutus*, *Cenococcum geophilum*, and *Rhizopogon roseolus* were able to use peptides and proteins as their sole N sources. In contrast, *Laccaria laccata* and *Lactarius rufus* had little ability to grow with peptides and proteins but they grew well with

**154**

N forms.

roots to absorb nutrients.

in nitrate-rich conditions [53].

their environment. Recently, an increasing number of studies showed that inorganic N enrichment in forest soils caused by pollution, fertilization or natural causes are leading to a reduction in the level of plant root colonization by ECM fungi, also shift fungal community in soils away from ECM fungi specialized in organic N acquisition to more generalist nitrophilic species and saprotrophs [70–72].

Other studies have concluded that differences in proteolytic activity between the species of ECM fungi could be explained by soil-derived selection pressures. For example, *Hebeloma crustuliniforme* expressed proteolytic activity in the presence of a readily available N source such as ammonium [73]. Ammonium has also been shown to repress the expression of amino-acid transporters and enzymes in N assimilation pathways in ECM fungi [74, 75]. The presence of inorganic N tightly down regulated soil organic matter degradation by *Paxillus involutus* as proved [76]. Such facts suggest that ECM fungal degradation activity would be controlled by environmental factors.

Different ECM species occupy different successional stages in forest development. This seems to be related to the proteolytic activity of fungi. When resource quality declines and organic matter accumulation declines during forest development, fungi with limited proteolytic activity is favored. For the cultivation of edible mushrooms, this means that we should pay attention to the natural preferences of the species for nitrogen uptake. This may concern especially the ECM species that are difficult to cultivate artificially.

#### *3.2.3 Fungal genetic characteristics determines the efficiency of N transition*

Recently, advances in genetics and molecular biological techniques have provided better understanding about nitrogen metabolism. The acquisition of inorganic N and the mineralization of organic N by ECM fungi have been proved by many molecular investigations. Ectomycorrhizal fungi encode a number of transporters to acquire nitrate and ammonium from soil, as well as a suite of enzymes and transporters necessary for utilizing organic N sources [77–79]. Ammonium importers such as AMT1, AMT2 and AMT3 have been functionally characterized in several ECM fungal species, such as, *Hebeloma cylindrosporum* [75, 80], *Tuber borchii* [81] and *Amanita muscaria* [82]. Nitrate transporters, such as LbNRT2 in *Laccaria bicolor* [83] and HcNRT2 in *H. cylindrosporum* [84], are also present in ECM genomes allowing N transport.

Ectomycorrhizal fungi have all evolved from their saprotrophic ancestors, and hence, ECM have the ability to decompose organic matter [85, 86]. The utilization of proteins by fungi requires the enzymatic degradation of proteins to peptides and amino acids before cellular uptake. Lindahl and Taylor [87] studied the genetic potential of ECM fungi to produce N-acetylhexosaminidases that hydrolyze chitin to N-acetylglucosamine. Thus, N-acetylglucosamine and amino acids replace ammonium and nitrate as the N sources [19].

Recently, the genomes of ECM fungi were found to contain the same or smaller number of copies of genes coding for secreted N and P targeting hydrolases than saprotrophs, pathogens, or ericoid mycorrhizal fungi [88]. This observation is surprising because the well-documented ability of ECM fungi to hydrolyze organic phosphate compounds and scavenge nitrogen through the degradation of proteins accumulated in litter. Miyauchi and colleagues [88] also showed that the ECM fungus *Paxillus involutus* was able, while assimilating organic N, to significantly modify organic matter with a free-radical-based mechanism similar to that of saprophytic brown-rot fungi [76]. Unlike the saprophytic fungi, *P. involutus* did not show any expression of genes encoding extracellular enzymes needed to metabolize the released C. This suggests that the degradation mechanism of this ECM fungus has evolved to assimilate organic N rather than C.

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*Conservation of Edible Ectomycorrhizal Mushrooms: Understanding of the ECM Fungi…*

to drive boreal forests towards a more severe N limitation at low N supply.

**4. Challenges in establishing edible ectomycorrhizal fungal culture** 

More than thousand species of ECM fungi produce edible mushrooms [95]. Some of them, such as *Amanita caesarea* (Scop.) Pers. *Boletus edulis* Bull., *Cantharellus cibarius* Fr. and *Tricholoma matsutake* (S. Ito and S. Imai) Singer, have economical value on international markets. The problem is that edible ECM fungi are usually more difficult to cultivate than saprophytic fungi because of the symbiotic relationship with a host tree is needed. In the past few decades, significant progress has been made in the cultivation of some fungi, such as *Lactarius deliciosus* (L.) Gray [96–98], *Lactarius hatsudake* Nobuj. Tanaka [99], *Suillus granulatus* (L.) Roussel [96], *Rhizopogon roseolus* (Corda) Th. Fr. [100], and *Lyophyllum shimeji* (Kawam.) Hongo [101]. In controlled conditions, however, the successful fruitbody or primordium formations are limited. Most of edible ECM fungi still cannot be cultivated. The major issues that need to be understood are the trophic relationships, biotic, edaphic, and climatic requirements for each mushroom. In this review, we focus on the nitrogen acquisition of edible ECM fungi for their mycelial culture and its effect on fruitbody formation. Secondly, we take *T. matsutake* as an example and discuss in detail about its ability to acquire nitrogen, its preferences, and possible strategies. Finally, we discuss about the further challenges – to conserve proper

We summarize the nitrogen sources used in mycelium culture and the cultivation experiments of edible ECM fungi in combination with ECM fungal morphological characteristics reported from the published studies (**Table 1**). As known, most edible ECM fungi are difficult for cultivation so far. We could get some hints for the ECM cultivation from experimentally observed nitrogen preferences

ECM fungi have diverse evolutionary origins and they use diverse decomposition mechanisms to access organic nitrogen entrapped in soil organic matter [91]. The timing and magnitude of decomposition activity seem to be controlled by the below-ground nitrogen quality and the above-ground carbon supply. Some ECM fungi might act as decomposers, not primarily to obtain C to their metabolism, but to search for organic N in the absence of readily available inorganic N [76, 92–94].

ECM fungi are able to breakdown soil organic N with differing efficiencies. It has been found that the uptake of amino acids by mycorrhizal fungi is related to the N content and carbon structure of the amino acid [89]. One hypothesis was proposed that the rate at which mycorrhizal fungi degrade large organic N polymers in soils is also controlled by the plant C resources available to the fungi to construct extracellular enzymes, as well as the bond strength and structural diversity of the target organic N compound although the direct tests of the hypothetical mechanism is still needed. Another study by Näsholm et al. [90] tested a model for C–N exchange between trees and mycorrhizal fungi. They found that ECM fungi transport smaller amounts of absorbed N to trees in N-limited than in N-rich conditions. The study found further that the greater allocation of C from trees to ECM fungi increases N retention into soil mycelium. The growth of these fungi is stimulated, and thus, N is immobilized and sequestered in soil. This mechanism was suggested

*3.2.4 ECM utilizing organic N in relation to receiving C from trees*

*DOI: http://dx.doi.org/10.5772/intechopen.95399*

**with fruitbody formation**

ecological conditions for edible ECM fungi to grow.

**4.1 Nitrogen sources in edible ECM fungal cultures**

*Conservation of Edible Ectomycorrhizal Mushrooms: Understanding of the ECM Fungi… DOI: http://dx.doi.org/10.5772/intechopen.95399*

#### *3.2.4 ECM utilizing organic N in relation to receiving C from trees*

*Nitrogen in Agriculture - Physiological, Agricultural and Ecological Aspects*

tion to more generalist nitrophilic species and saprotrophs [70–72].

*3.2.3 Fungal genetic characteristics determines the efficiency of N transition*

Recently, advances in genetics and molecular biological techniques have provided better understanding about nitrogen metabolism. The acquisition of inorganic N and the mineralization of organic N by ECM fungi have been proved by many molecular investigations. Ectomycorrhizal fungi encode a number of transporters to acquire nitrate and ammonium from soil, as well as a suite of enzymes and transporters necessary for utilizing organic N sources [77–79]. Ammonium importers such as AMT1, AMT2 and AMT3 have been functionally characterized in several ECM fungal species, such as, *Hebeloma cylindrosporum* [75, 80], *Tuber borchii* [81] and *Amanita muscaria* [82]. Nitrate transporters, such as LbNRT2 in *Laccaria bicolor* [83] and HcNRT2 in *H. cylindrosporum* [84], are also present in

Ectomycorrhizal fungi have all evolved from their saprotrophic ancestors, and hence, ECM have the ability to decompose organic matter [85, 86]. The utilization of proteins by fungi requires the enzymatic degradation of proteins to peptides and amino acids before cellular uptake. Lindahl and Taylor [87] studied the genetic potential of ECM fungi to produce N-acetylhexosaminidases that hydrolyze chitin to N-acetylglucosamine. Thus, N-acetylglucosamine and amino acids replace

Recently, the genomes of ECM fungi were found to contain the same or smaller number of copies of genes coding for secreted N and P targeting hydrolases than saprotrophs, pathogens, or ericoid mycorrhizal fungi [88]. This observation is surprising because the well-documented ability of ECM fungi to hydrolyze organic phosphate compounds and scavenge nitrogen through the degradation of proteins accumulated in litter. Miyauchi and colleagues [88] also showed that the ECM fungus *Paxillus involutus* was able, while assimilating organic N, to significantly modify organic matter with a free-radical-based mechanism similar to that of saprophytic brown-rot fungi [76]. Unlike the saprophytic fungi, *P. involutus* did not show any expression of genes encoding extracellular enzymes needed to metabolize the released C. This suggests that the degradation mechanism of this ECM fungus

are difficult to cultivate artificially.

ECM genomes allowing N transport.

ammonium and nitrate as the N sources [19].

has evolved to assimilate organic N rather than C.

their environment. Recently, an increasing number of studies showed that inorganic N enrichment in forest soils caused by pollution, fertilization or natural causes are leading to a reduction in the level of plant root colonization by ECM fungi, also shift fungal community in soils away from ECM fungi specialized in organic N acquisi-

Other studies have concluded that differences in proteolytic activity between the species of ECM fungi could be explained by soil-derived selection pressures. For example, *Hebeloma crustuliniforme* expressed proteolytic activity in the presence of a readily available N source such as ammonium [73]. Ammonium has also been shown to repress the expression of amino-acid transporters and enzymes in N assimilation pathways in ECM fungi [74, 75]. The presence of inorganic N tightly down regulated soil organic matter degradation by *Paxillus involutus* as proved [76]. Such facts suggest that ECM fungal degradation activity would be controlled by environmental factors. Different ECM species occupy different successional stages in forest development. This seems to be related to the proteolytic activity of fungi. When resource quality declines and organic matter accumulation declines during forest development, fungi with limited proteolytic activity is favored. For the cultivation of edible mushrooms, this means that we should pay attention to the natural preferences of the species for nitrogen uptake. This may concern especially the ECM species that

**156**

ECM fungi are able to breakdown soil organic N with differing efficiencies. It has been found that the uptake of amino acids by mycorrhizal fungi is related to the N content and carbon structure of the amino acid [89]. One hypothesis was proposed that the rate at which mycorrhizal fungi degrade large organic N polymers in soils is also controlled by the plant C resources available to the fungi to construct extracellular enzymes, as well as the bond strength and structural diversity of the target organic N compound although the direct tests of the hypothetical mechanism is still needed. Another study by Näsholm et al. [90] tested a model for C–N exchange between trees and mycorrhizal fungi. They found that ECM fungi transport smaller amounts of absorbed N to trees in N-limited than in N-rich conditions. The study found further that the greater allocation of C from trees to ECM fungi increases N retention into soil mycelium. The growth of these fungi is stimulated, and thus, N is immobilized and sequestered in soil. This mechanism was suggested to drive boreal forests towards a more severe N limitation at low N supply.

ECM fungi have diverse evolutionary origins and they use diverse decomposition mechanisms to access organic nitrogen entrapped in soil organic matter [91]. The timing and magnitude of decomposition activity seem to be controlled by the below-ground nitrogen quality and the above-ground carbon supply. Some ECM fungi might act as decomposers, not primarily to obtain C to their metabolism, but to search for organic N in the absence of readily available inorganic N [76, 92–94].

#### **4. Challenges in establishing edible ectomycorrhizal fungal culture with fruitbody formation**

More than thousand species of ECM fungi produce edible mushrooms [95]. Some of them, such as *Amanita caesarea* (Scop.) Pers. *Boletus edulis* Bull., *Cantharellus cibarius* Fr. and *Tricholoma matsutake* (S. Ito and S. Imai) Singer, have economical value on international markets. The problem is that edible ECM fungi are usually more difficult to cultivate than saprophytic fungi because of the symbiotic relationship with a host tree is needed. In the past few decades, significant progress has been made in the cultivation of some fungi, such as *Lactarius deliciosus* (L.) Gray [96–98], *Lactarius hatsudake* Nobuj. Tanaka [99], *Suillus granulatus* (L.) Roussel [96], *Rhizopogon roseolus* (Corda) Th. Fr. [100], and *Lyophyllum shimeji* (Kawam.) Hongo [101]. In controlled conditions, however, the successful fruitbody or primordium formations are limited. Most of edible ECM fungi still cannot be cultivated. The major issues that need to be understood are the trophic relationships, biotic, edaphic, and climatic requirements for each mushroom. In this review, we focus on the nitrogen acquisition of edible ECM fungi for their mycelial culture and its effect on fruitbody formation. Secondly, we take *T. matsutake* as an example and discuss in detail about its ability to acquire nitrogen, its preferences, and possible strategies. Finally, we discuss about the further challenges – to conserve proper ecological conditions for edible ECM fungi to grow.

#### **4.1 Nitrogen sources in edible ECM fungal cultures**

We summarize the nitrogen sources used in mycelium culture and the cultivation experiments of edible ECM fungi in combination with ECM fungal morphological characteristics reported from the published studies (**Table 1**). As known, most edible ECM fungi are difficult for cultivation so far. We could get some hints for the ECM cultivation from experimentally observed nitrogen preferences


**159**

**ECM fungi**

*L. lacata*

orgN

NH4

+ (poor on

orgN)

*L. bicolor*NH4

(poor on NO3

or orgN)

NH4

+, NO3

on amino acid,

good on urea)

NH4

+ (poor on

orgN)

**Lactarius** *L. deliciosus*

*L. rufus* **Lyophyllus**

*L. shimeji*

**Paxillus** *P. involutus* **Scleroderma**

*S. citrinum*

**Suillus** *S. bovinus*

NH4

+ or orgN

orgN

NH4

+ and orgN

[115]

Ho

[113]

[112]

Ho Ho

> Forest soil

> [116]

Long

Long

8.2 ± 0.7 (17)

[102]

Long

7.1 ± 0.7 (7)

[102]

NH4

+ plus orgN orgN (a variable

among strains)

− (poor

−

,

+

**Mycelium growth**

**Mycorrhization**

**Fruitbody formation**

**Ref** [110] [112]

[113] [114]

[67]

Hi

[98]

[67]

Contact/

4.2 ± 0.3 (54)

4.3 ± 0.5 (3)

[111]

[102]

Medium-smooth

**Hydrophobicity**

**Exploration type**

**δ15N (‰)** 

**Ref.**

**(Mean ± SD) (n)**

3.0 ± 0.4 (3) cap

[111]

*Conservation of Edible Ectomycorrhizal Mushrooms: Understanding of the ECM Fungi…*

*DOI: http://dx.doi.org/10.5772/intechopen.95399*

1.8 (1) cap

[111]

#### *Nitrogen in Agriculture - Physiological, Agricultural and Ecological Aspects*


#### *Conservation of Edible Ectomycorrhizal Mushrooms: Understanding of the ECM Fungi… DOI: http://dx.doi.org/10.5772/intechopen.95399*

**ECM fungi**

*L. lacata*

orgN

NH4

+ (poor on

orgN)

*L. bicolor*NH4

(poor on NO3

or orgN)

NH4

+, NO3

on amino acid,

good on urea)

NH4

+ (poor on

orgN)

**Lactarius** *L. deliciosus*

*L. rufus* **Lyophyllus**

*L. shimeji*

**Paxillus** *P. involutus* **Scleroderma**

*S. citrinum*

**Suillus** *S. bovinus*

NH4

+ or orgN

orgN

NH4

+ plus orgN orgN (a variable

among strains)

− (poor

−

,

+

**Mycelium growth**

*Nitrogen in Agriculture - Physiological, Agricultural and Ecological Aspects*

**158**

**ECM fungi**

**Amanita** *A. caesarea*

**Boletus** *B. edulis* *B. reticulatus*

*Boletus* sp. **Cantharellus**

*C. cibarius* **Cortinarius** *C. variecolor*

**Hebeloma**

*H.* 

*cylindrosporum*

*H. radicosum* *Hebeloma* sp.

**Hydnum** *H. repandum*

**Laccaria**

NO3

(poor on NH4

+

)

− or ON

NH4

+ (poor on

orgN)

orgN NH4

NH4

+ and orgN

[107]

Hi

[48]

Ho

> orgN

orgN (but a variable

[59]

among strains)

ON NH4

orgN

[108]

Ho

[109]

Hi

Short

0.5 ± 0.6 (15)

[102]

Medium-fringe

12 (1) cap

[102]

[108]

+

[57]

[67]

Ho

Short/

*2.7 ± 1.1 (7)*

[102]

medium-fringe

Medium-fringe

6.8 ± 0.3 (100)

[102]

4.3 ± 1.4 (8)

[102]

+ and orgN

[106]

[105]

NH4

+ (poor on

orgN)

**Mycelium** 

**Mycorrhization**

**Fruitbody formation**

**Ref**

**Hydrophobicity**

Hi

[103]

Ho

Long

5.8 ± 1.0 (17)

8.66 (1)

[104]

[102]

**Exploration** 

**δ15N (‰)** 

**Ref.**

**(Mean ± SD) (n)**

**type**

Medium-smooth

3.1 ± 0.5 (35)

[102]

**growth**


*Nitrogen in Agriculture - Physiological, Agricultural and Ecological Aspects*
