Open access peer-reviewed chapter

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

Written By

Lu-Min Vaario and Norihisa Matsushita

Submitted: 20 September 2020 Reviewed: 07 December 2020 Published: 29 January 2021

DOI: 10.5772/intechopen.95399

From the Edited Volume

Nitrogen in Agriculture - Physiological, Agricultural and Ecological Aspects

Edited by Takuji Ohyama and Kazuyuki Inubushi

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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.


  • 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 well-growing 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, 9, 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].

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, 20, 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, 36, 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]. 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 non-humic, 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, 57, 58, 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 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 δ15N 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 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, 71, 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, 78, 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.

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

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, 93, 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, 97, 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 and mycorrhizal formation. In pure culture conditions, most of the studied fungi appeared to favor ammonium N. Some species, namely Amanita caesarea, Cantharellus cibarius, Lactarius bicolor, Suillus variegatus were not able to grow nitrate as the sole N source [48, 103, 113].

ECM fungiMycelium growthMycorrhizationFruitbody formationRefHydrophobicityExploration typeδ15N (‰) (Mean ± SD) (n)Ref.
AmanitaHiMedium-smooth3.1 ± 0.5 (35)[102]
A. caesareaNH4+ (poor on orgN)[103]
BoletusHoLong5.8 ± 1.0 (17)[102]
8.66 (1)[104]
B. edulisorgN[105]
B. reticulatusNH4+ and orgN[106]
Boletus sp.NH4+ and orgN[107]
CantharellusHi4.3 ± 1.4 (8)[102]
C. cibariusNH4+ (poor on orgN)[48]
CortinariusHoMedium-fringe6.8 ± 0.3 (100)[102]
C. variecolororgN[67]
HebelomaHoShort/medium-fringe2.7 ± 1.1 (7)[102]
H. cylindrosporumorgN (but a variable among strains)[59]
H. radicosumNH4+[108]
Hebeloma sp.orgN[108]
HydnumHoMedium-fringe12 (1) cap[102]
H. repandumNO3 or ON (poor on NH4+)[109]
LaccariaHiShort0.5 ± 0.6 (15)[102]
L. lacataorgN[110]3.0 ± 0.4 (3) cap[111]
NH4+ (poor on orgN)[112]
L. bicolorNH4+ (poor on NO3, or orgN)[113]1.8 (1) cap[111]
NH4+, NO3 (poor on amino acid, good on urea)[114]
NH4+ (poor on orgN)[67]
LactariusHiContact/Medium-smooth4.2 ± 0.3 (54)[102]
4.3 ± 0.5 (3)[111]
L. deliciosusNH4+ plus orgN[98]
L. rufusorgN (a variable among strains)[67]
L. shimejiNH4+ and orgN[115]
PaxillusHoLong7.1 ± 0.7 (7)[102]
P. involutusorgN[113]
S. citrinumNH4+ or orgN[112]HoLong
SuillusHoLong8.2 ± 0.7 (17)[102]
S. bovinusForest soil[116]
S. lutusNH4+ (poor on orgN)[112]
S. variegatusorgN[113]5.7 ± 1.1 (4) cap[111]
TricholomaHoMedium-fringe9.3 ± 0.6 (35)[102]
T. imbricatumNH4+ or NO3 or orgN (gained better grwoth in iorgN)[117]
T. bakamatsutakeNH4+ or orgN (poor on NO3)[118]
T. matsutakeNH4+ pluse orgN[119]16.8 ± 2.3 (15)[120]
orgN (sustaining symbiotic relationship)[122]
T. terreumorgN (gained better growth)[123]
TuberHiShort15.1 ± 0.6 (9)[102]
T. sinenseorgN( gained better growth)[124]

Table 1.

Fungal growth, symbiosis and fruitbody formation observed using different nitrogen sources in edible ectomycorrhizal fungi in combination with the information of hydrophobicity, exploration type and δ15N of the fruitbodies.

* NH4+, ammonum nitrogen; NO3, nitrate nitrogen; orgN, organic nitrogen. Ho, hydrophobic; Hi, hydrophibic.

However, many of the edible ECM fungi, namely Amanita caesarea [103, 105], Cantharellus cibarius [48], Cortinarius variecolor [67], Paxillus involutus, Suillus variegatus [113], Tricholoma terreum [123], and Tuber sinense [124] were able to grow on the media containing organic N (protein) as the sole nitrogen. Moreover, some fungi belonging to Lactarius genus had limited capacity to utilize protein N [113, 114]. Hebeloma cylindrosporum was able to experimentally utilize a wide range of amino acids and other simple (e.g. urea) or complex (e.g. proteins) compounds [6, 59].

The studied forms of N often predominate soil solution and the culturing results might be assumed to hold true in nature. However, it is worth mentioning is that the optimal nitrogen in the mycelium culture does not necessarily reflect the nitrogen preference of the ECM fungus under natural conditions because environmental factors affect. This was shown with H. cylindrosporum growing in nature. Wild dikaryotic strains of H. cylindrosporum isolated from two different habitat types had different N preferences [6].

Cultivation of edible ECM mushrooms has been successful in cases of two truffles Tuber melanosporum Vittad. and Tuber aestivum Vittad. They are cultivated commercially around the world [125]. In addition, some success has been achieved with Lactarius deliciosus [126, 127] and Boletus edulis [128]. Regarding truffle production, it has been suggested that most soils contain enough N to maintain both fungal and tree growth [125]. Similarly, Lactarius deliciosus was cultivated experimentally in forest soil, which was observed to meet the demands for fruitbody formation [126]. It has also been demonstrated that the nutritional properties of soil and the forestry history the natural development of ECM mushrooms in forest ecosystems [129]. A productive and diverse ECM mushroom community resembling natural communities developed when abandoned farmland in Mediterranean dry area was forested with Pinus sp.

In summary, productive ECM community can grow in natural soils. However, the challenges faced in artificial cultivation has not been solved.

4.2 Nitrogen source requirements for Tricholoma matsutake mycelial culture and mycorrhizal synthesis

Tricholoma matsutake is among the most economically valuable mushrooms in the world. Its taxonomy, distribution, ecology, physiology, and cultivation has been studied widely [130]. Here, we summarize the key results linking matsutake ecological characteristics and nutrient requirements focusing on nitrogen.

Matsutake colonizes the roots of its host trees via an ECM association (Figure 1a and b). It develops an extraradical mycelium in the rhizosphere and in the surrounding soil area. This can be seen as a white rhizosphere area and it corresponds to the mycelium-soil aggregated zone, called a shiro [131] (Figure 1c and d). Matsutake shiro grows in the form of a concentric or horseshoe-like circle, depending on the rhizosphere conditions, around the host plant at the rate of approximately 10–15 cm per year [131, 132]. The production of matsutake mushrooms changes periodically. Based on our field observations, the part of mycorrhizal root tips is degraded prior to matsutake fruiting. The extraradical mycelium might grow towards new roots and colonizing. Such a hyphal growth strategy indicates that matsutake symbiosis may often need to be renewed and form new mycorrhizas to acquire nutrients (data not published). Among the mycorrhizal associations, such a phenomenon does not seem to be rare. Hortal and colleagues [133] found that the plant had the ability to limit the root tip colonization of the least cooperative symbiont, and therefore, influence the outcome of ECM fungi competition. Such reduction in colonization did not result in a reduction in carbon allocation to the fungus providing the lowest amount of nitrogen.

Figure 1.

The ectomycorrhizal edible mushroom—Tricholoma matsutake (a) the root of Pinus sylvestris seedling is colonized by T. matsutake fungal mycelium, forms mycorrhizas; (b) the transverse section of ECM root showing the Hartig net (hn) development within the cortex; (c) matsutake mushrooms form in a conifer mixed forest in southern of Finland; (d) the matsutake shiro (arrow) after the mushrooms be harvested (photos were taken by Lu Min Vaario).

It is worth noting that decayed mycorrhizal roots together with mycelium-soil aggregated zone might be important organic nutrient sources for matsutake. Recently, the natural abundance of isotopes data showed a very high δ15N value in T. matsutake fruitbodies, which were sampled from Finland and Japan [120]. Matsutake usually grow at B layer of mineral soil [131], such taxa obtain their N could explain for high δ15N values (see review [102]). More importantly, the high δ15N value in matsutake is an indicator of organic N uptake from soil because the great variation of 15N content observed among ECM taxa has been reported to be related to the differences in organic N utilization [111]. In addition, a literature study shows that mycorrhizal taxa with proteolytic activities generally show high δ15N values [67]. Therefore, we conclude that matsutake has a greater proteolytic activity to digest chemically complex 15N-enriched organic matter in soil during matsutake fruitbody development.

In addition to proteases, matsutake produces organic matter degradation enzymes such as acid proteinase [134, 135] and β-glucosidase [136]. Relatively high enzyme activities, β-glucosidase and xylosidase, were detected from matsutake cultures in vitro and in shiro soil [137, 138]. The genome of T. matsutake encodes two GH7 cellobiohydrolases [88], which is in agreement with its known facultative saprotrophic activity [136, 138]. However, no further evidence of any strong saprotrophic characteristics of matsutake was found. It could be speculated that these ECM fungi produce certain levels of carbohydrase, not to fully degrade organic matter to access C but N. Kawai and Abe [121] reported that dried beer yeast, corn steep liquor, casein hydrolysate, and polypeptone were good N sources for matsutake mycelium culture whereas nitrate was not. Dry beer yeast (Ebios, Asahi Beer Inc., Tokyo, Japan), as the sole N source, showed promising matsutake mycelium growth and as well mycorrhizal formation [139] (personal communication with Dr. A. Yamada).

Several agar media such as MMN, MNC, Hamada containing both inorganic and organic N are widely used to culture the mycelium of T. matsutake [119, 140]. However, the question whether matsutake prefers organic nitrogen is worth of considering. Usually, more inorganic N than organic N is present in the soil top layer. Some studies suggested that increased N deposition could reduce fruitbody production [141]. Nohrstedt [142] reported a 30% decrease in sporocarp production by Cantharellus cibarius in a central Swedish pine forest after the application of 150 kg N ha−1 ammonium nitrate. The presence of nitrate ions has been shown to have negative effects on the development of some ECM fungi both in vitro and in soil [143, 144]. Removal of the litter layer has been considered an important method to improve the productivity of matsutake in many Asian countries [145]. It has also been shown that the removal of the upper organic soil layers of the forest floor can improve the sporocarp production of some other ECM fungi [146, 147]. The explanation might be that competition with other microbes diminishes. Litter and organic soil provide carbon and nutrients for microbes, especially for saprotrophic fungi that would compete with T. matsutake in the shiro [148].

4.3 Research prospects

Cultivation of ectomycorrhizal mushrooms is still facing many challenges. Although some species of ECM fungi can form the primordium of fruiting bodies on several media, they usually do not develop further into mature fruiting bodies. So far, the most successful efforts have been carried out with the mycorrhizal plants growing in soil. Soil nutrients and soil microbial communities together with climatic factors have shown to affect significantly the persistence of ectomycorrhizas in outplanted inoculated plants, and further, the successful fruiting. The observed suppression of many mycorrhizal mushrooms has been linked to indirect effects of air pollution, in particular to increases in nitrogen deposition accumulating into litter and humus [149, 150]. A thorough understanding of the ecological and environmental factors regulating the ECM fungal species is a prerequisite for their cultivation.

Ectomycorrhizal fungi colonize the roots of their host plants and improve plants’ access to nutrients, especially nitrogen. In exchange, host plants deliver a significant portion of their photosynthesized carbon to the ECM fungi. However, we need more accurate understanding of the ECM fungi mediated C and N movement within forest ecosystems. ECM fungi may follow a similar pattern with the amount of C delivered being related to the amount of N sourced by the fungus [77, 151], although this is still controversial [133]. Production of ECM mushrooms do need a balanced nutrient either assimilating by ECM fungi or by other soil microbial.

It has been suggested that the growth of ECM fungi and the formation of mycorrhizas are promoted by certain mycorrhizosphere bacteria, termed ‘mycorrhizal helper bacteria’ [152]. Some mycorrhizal fungi-associated bacteria are also known to fix nitrogen [153, 154]. However, there is still no evidence that the fungus would directly benefit from its associated bacteria. Sporocarps of Cantharellus cibarius contain large amount of bacteria, in particular fluorescent Pseudomonas [155]. Some species of bacteria such as Streptomyces spp., Paenibacillus spp. and Bacillales spp. were isolated from the mycorrhizal root tips and fruitbodies of T. matsutake as well [156, 157, 158]. Otherwise, the information about mycorrhizas-associated bacteria and their effect on the nutrient uptake of ECM fungi is limited. These studies, however, hint that the production of ectomycorrhizal mushrooms may require teamwork to obtain enough nutrients from the environment.

In conclusion, ECM fungi play an important role in the nutrient cycle of forest ecosystems, especially on mediating C and N movement. A better understanding of the nitrogen status of the habitat of ECM fungi, nutrients movements within the ecosystems, as well as the ECM fungal hyphal structures should be the first step for cultivation of ECM edible mushrooms. The methodological advances in these areas in combination with forest management may allow the successful establishment of commercial plantations and production of edible ECM mushrooms in forests.


  1. 1. Pilz D, Molina R. Managing forest ecosystems to conserve fungus diversityandsustain wild mushroomharvests. US Department of Agriculture, Forest Service, Pacific Northwest Research Station, 1996
  2. 2. Führer E. Forest functions, ecosystem stability and management. For Ecol Manage 2000; 132: 29-38
  3. 3. Mandzak JM, Moore JA. The role of nutrition in the health of inland western forests. J Sustain For 1994; 2: 191-210
  4. 4. O’Neill KP. Soils as an indicator of forest health: a guide to the collection, analysis, and interpretation of soil indicator data in the Forest Inventory and Analysis program. USDA Forest Service, North Central Research Station, 2005
  5. 5. Schimel JP, Bennett J. Nitrogen mineralization: challenges of a changing paradigm. Ecology 2004; 85: 591-602
  6. 6. Abuarghub SM, Read DJ. 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 Phytol 1988; 108: 425-431
  7. 7. Kelley KR, Stevenson FJ. Forms and nature of organic N in soil. In: Nitrogen Economy in Tropical Soils. Springer, 1995, pp. 1-11
  8. 8. Hartley J, Cairney JWG, Meharg AA. Do ectomycorrhizal fungi exhibit adaptive tolerance to potentially toxic metals in the environment? Plant Soil 1997; 189: 303-319
  9. 9. Bandou E, Lebailly F, Muller F, et al. The ectomycorrhizal fungus Scleroderma bermudense alleviates salt stress in seagrape (Coccoloba uvifera L.) seedlings. Mycorrhiza 2006; 16: 559-565
  10. 10. Nadeem SM, Ahmad M, Zahir ZA, et al. The role of mycorrhizae and plant growth promoting rhizobacteria (PGPR) in improving crop productivity under stressful environments. Biotechnol Adv 2014; 32: 429-448
  11. 11. Hall IR, Yun W, Amicucci A. Cultivation of edible ectomycorrhizal mushrooms. TRENDS Biotechnol 2003; 21: 433-438
  12. 12. Boa E. Non-wood forest products. Wild edible fungi a glob overview of their use and importance to people. 2004 FAO
  13. 13. Smith SE. Read. DJ 1997. Mycorrhizal Symbiosis. Academic Press London
  14. 14. Landeweert R, Hoffland E, Finlay RD, et al. Linking plants to rocks: ectomycorrhizal fungi mobilize nutrients from minerals. Trends Ecol Evol 2001; 16: 248-254
  15. 15. Phillips RP, Brzostek E, Midgley MG. The mycorrhizal-associated nutrient economy: a new framework for predicting carbon–nutrient couplings in temperate forests. New Phytol 2013; 199: 41-51
  16. 16. Brundrett MC. Mycorrhizal associations and other means of nutrition of vascular plants: understanding the global diversity of host plants by resolving conflicting information and developing reliable means of diagnosis. Plant Soil 2009; 320: 37-77
  17. 17. Rinaldi AC, Comandini O, Kuyper TW. Ectomycorrhizal fungal diversity: seperating the wheat from the chaff. Fungal Divers 2008; 33: 1-45
  18. 18. Tedersoo L, Mett M, Ishida TA, et al. Phylogenetic relationships among host plants explain differences in fungal species richness and community composition in ectomycorrhizal symbiosis. New Phytol 2013; 199: 822-831
  19. 19. Read DJ, Perez-Moreno J. Mycorrhizas and nutrient cycling in ecosystems–a journey towards relevance? New Phytol 2003; 157: 475-492
  20. 20. Orwin KH, Kirschbaum MUF, St John MG, et al. Organic nutrient uptake by mycorrhizal fungi enhances ecosystem carbon storage: a model-based assessment. Ecol Lett 2011; 14: 493-502
  21. 21. Zak DR, Pellitier PT, Argiroff W, et al. Exploring the role of ectomycorrhizal fungi in soil carbon dynamics. New Phytol 2019; 223: 33-39
  22. 22. Toljander JF, Eberhardt U, Toljander YK, et al. Species composition of an ectomycorrhizal fungal community along a local nutrient gradient in a boreal forest. New Phytol 2006; 170: 873-884
  23. 23. Lin G, McCormack ML, Ma C, et al. Similar below-ground carbon cycling dynamics but contrasting modes of nitrogen cycling between arbuscular mycorrhizal and ectomycorrhizal forests. New Phytol 2017; 213: 1440-1451
  24. 24. Laliberté E, Lambers H, Burgess TI, et al. Phosphorus limitation, soil-borne pathogens and the coexistence of plant species in hyperdiverse forests and shrublands. New Phytol 2015; 206: 507-521
  25. 25. Egerton-Warburton LM, Johnson NC, Allen EB. Mycorrhizal community dynamics following nitrogen fertilization: a cross-site test in five grasslands. Ecol Monogr 2007; 77: 527-544
  26. 26. Steidinger BS, Crowther TW, Liang J, et al. Climatic controls of decomposition drive the global biogeography of forest-tree symbioses. Nature 2019; 569: 404-408
  27. 27. Smith ML, Bruhn JN, Anderson JB. The fungus Armillaria bulbosa is among the largest and oldest living organisms. Nature 1992; 356: 428-431
  28. 28. Agerer R. Exploration types of ectomycorrhizae. Mycorrhiza 2001; 11: 107-114
  29. 29. Unestam T, Sun Y-P. Extramatrical structures of hydrophobic and hydrophilic ectomycorrhizal fungi. Mycorrhiza 1995; 5: 301-311
  30. 30. Hobbie EA, Agerer R. Nitrogen isotopes in ectomycorrhizal sporocarps correspond to belowground exploration types. Plant Soil 2010; 327: 71-83
  31. 31. Finlay RD, Read DJ. The structure and function of the vegetative mycelium of ectomycorrhizal plants: II. The uptake and distribution of phosphorus by mycelial strands interconnecting host plants. New Phytol 1986; 103: 157-165
  32. 32. Burgess T, Dell B, Malajczuk N. Variation in mycorrhizal development and growth stimulation by 20 Pisolithus isolates inoculated on to Eucalyptus grandis W. Hill ex Maiden. New Phytol 1994; 127: 731-739
  33. 33. Burgess TI, Malajczuk N, Grove TS. The ability of 16 ectomycorrhizal fungi to increase growth and phosphorus uptake of Eucalyptus globulus Labill. and E. diversicolor F. Muell. Plant Soil 1993; 153: 155-164
  34. 34. Dell B, Malajczuk N, Bougher NL, et al. Development and function of Pisolithus and Scleroderma ectomycorrhizas formed in vivo with Allocasuarina, Casuarina and Eucalyptus. Mycorrhiza 1994; 5: 129-138
  35. 35. Colpaert J V, Van Assche JA, Luijtens K. The growth of the extramatrical mycelium of ectomycorrhizal fungi and the growth response of Pinus sylvestris L. New Phytol 1992; 120: 127-135
  36. 36. Islam MR, Tudryn G, Bucinell R, et al. Morphology and mechanics of fungal mycelium. Sci Rep 2017; 7: 1-12
  37. 37. Thomson BD, Grove TS, Malajczuk N, et al. The effectiveness of ectomycorrhizal fungi in increasing the growth of Eucalyptus globulus Labill. in relation to root colonization and hyphal development in soil. New Phytol 1994; 126: 517-524
  38. 38. Cairney JWG. Extramatrical mycelia of ectomycorrhizal fungi as moderators of carbon dynamics in forest soil. Soil Biol Biochem 2012; 47: 198-208
  39. 39. Vestgarden LS. Carbon and nitrogen turnover in the early stage of Scots pine (Pinus sylvestris L.) needle litter decomposition: effects of internal and external nitrogen. Soil Biol Biochem 2001; 33: 465-474
  40. 40. Wu T. Can ectomycorrhizal fungi circumvent the nitrogen mineralization for plant nutrition in temperate forest ecosystems? Soil Biol Biochem 2011; 43: 1109-1117
  41. 41. Alexander IJ. The significance of ectomycorrhizas in the nitrogen cycle. In’Nitrogen as an Ecological Factor’. (Eds J. A. Lee, S. McNeill and IH Rorison.).1983; pp. 69-94
  42. 42. Schnitzer M, Schulten H-R. New ideas on the chemical make-up of soil humic and fulvic acids. Futur Prospect soil Chem 1998; 55: 153-177
  43. 43. Jones DL, Kielland K. Soil amino acid turnover dominates the nitrogen flux in permafrost-dominated taiga forest soils. Soil Biol Biochem 2002; 34: 209-219
  44. 44. Sowden FJ, Chen Y, Schnitzer M. The nitrogen distribution in soils formed under widely differing climatic conditions. Geochim Cosmochim Acta 1977; 41: 1524-1526
  45. 45. Werdin-Pfisterer NR, Kielland K, Boone RD. Soil amino acid composition across a boreal forest successional sequence. Soil Biol Biochem 2009; 41: 1210-1220
  46. 46. Vancampenhout K, Wouters K, De Vos B, et al. Differences in chemical composition of soil organic matter in natural ecosystems from different climatic regions–A pyrolysis–GC/MS study. Soil Biol Biochem 2009; 41: 568-579
  47. 47. Meyer FH. Distribution of ectomycorrhizae in native and man-made forests. Ectomycorrhizae their Ecol Physiol 1973; 79-105
  48. 48. Rangel-Castro IJ, Danell E, Taylor AF. Use of different nitrogen sources by the edible ectomycorrhizal mushroom Cantharellus cibarius. Mycorrhiza 2002; 12: 131-137
  49. 49. Chalot M, Plassard C. Ectomycorrhiza and nitrogen provision to the host tree. Ecol Asp nitrogen Metab plants 2011; 69-94
  50. 50. Clemmensen KE, Sorensen PL, Michelsen A, et al. Site-dependent N uptake from N-form mixtures by arctic plants, soil microbes and ectomycorrhizal fungi. Oecologia 2008; 155: 771-783
  51. 51. Nygren CMR, Eberhardt U, Karlsson M, et al. Growth on nitrate and occurrence of nitrate reductase-encoding genes in a phylogenetically diverse range of ectomycorrhizal fungi. New Phytol 2008; 180: 875-889
  52. 52. Turnbull MH, Goodall R, Stewart GR. The impact of mycorrhizal colonization upon nitrogen source utilization and metabolism in seedlings of Eucalyptus grandis Hill ex Maiden and Eucalyptus maculata Hook. Plant Cell Environ 1995; 18: 1386-1394
  53. 53. Hobbie EA, Colpaert J V, White MW, et al. Nitrogen form, availability, and mycorrhizal colonization affect biomass and nitrogen isotope patterns in Pinus sylvestris. Plant Soil 2008; 310: 121
  54. 54. Read DJ, Leake JR, Perez-Moreno J. Mycorrhizal fungi as drivers of ecosystem processes in heathland and boreal forest biomes. Can J Bot 2004; 82: 1243-1263
  55. 55. Melin E, Nilsson H. Transfer of labelled nitrogen from glutamic acid to pine seedlings through the mycelium of Boletus variegatus (Sw.) Fr. Nature 1953; 171: 134
  56. 56. Chalot M, Brun A. Physiology of organic nitrogen acquisition by ectomycorrhizal fungi and ectomycorrhizas. FEMS Microbiol Rev 1998; 22: 21-44
  57. 57. Plassard C, Bonafos B, Touraine B. Differential effects of mineral and organic N sources, and of ectomycorrhizal infection by Hebeloma cylindrosporum, on growth and N utilization in Pinus pinaster. Plant Cell Environ 2000; 23: 1195-1205
  58. 58. Taylor AFS, Fransson PM, Högberg P, et al. Species level patterns in 13C and 15N abundance of ectomycorrhizal and saprotrophic fungal sporocarps. New Phytol 2003; 159: 757-774
  59. 59. Guidot A, Verner M-C, Debaud J-C, et al. Intraspecific variation in use of different organic nitrogen sources by the ectomycorrhizal fungus Hebeloma cylindrosporum. Mycorrhiza 2005; 15: 167-177
  60. 60. Abuzinadah RA, Read DJ. The role of proteins in the nitrogen nutrition of ectomycorrhizal plants: I. Utilization of peptides and proteins by ectomycorrhizal fungi. New Phytol 1986; 103: 481-493
  61. 61. Courty P, Pritsch K, Schloter M, et al. Activity profiling of ectomycorrhiza communities in two forest soils using multiple enzymatic tests. New Phytol 2005; 167: 309-319
  62. 62. Lilleskov EA, Hobbie EA, Horton TR. Conservation of ectomycorrhizal fungi: exploring the linkages between functional and taxonomic responses to anthropogenic N deposition. fungal Ecol 2011; 4: 174-183
  63. 63. Peay KG, Kennedy PG, Bruns TD. Rethinking ectomycorrhizal succession: are root density and hyphal exploration types drivers of spatial and temporal zonation? fungal Ecol 2011; 4: 233-240
  64. 64. Dawson TE, Mambelli S, Plamboeck AH, et al. Stable isotopes in plant ecology. Annu Rev Ecol Syst 2002; 33: 507-559
  65. 65. Hobbie EA, Hobbie JE. Natural abundance of 15 N in nitrogen-limited forests and tundra can estimate nitrogen cycling through mycorrhizal fungi: a review. Ecosystems 2008; 11: 815-830
  66. 66. Trudell SA, Rygiewicz PT, Edmonds RL. Patterns of nitrogen and carbon stable isotope ratios in macrofungi, plants and soils in two old-growth conifer forests. New Phytol 2004; 164: 317-335
  67. 67. Lilleskov EA, Hobbie EA, Fahey TJ. Ectomycorrhizal fungal taxa differing in response to nitrogen deposition also differ in pure culture organic nitrogen use and natural abundance of nitrogen isotopes. New Phytol 2002; 154: 219-231
  68. 68. Hobbie EA, Sánchez FS, Rygiewicz PT. Controls of isotopic patterns in saprotrophic and ectomycorrhizal fungi. Soil Biol Biochem 2012; 48: 60-68
  69. 69. Kranabetter JM. Ectomycorrhizal fungi and the nitrogen economy of conifers—implications for genecology and climate change mitigation. Botany 2014; 92: 417-423
  70. 70. Pardo LH, Fenn ME, Goodale CL, et al. Effects of nitrogen deposition and empirical nitrogen critical loads for ecoregions of the United States. Ecol Appl 2011; 21: 3049-3082
  71. 71. Morrison EW, Frey SD, Sadowsky JJ, et al. Chronic nitrogen additions fundamentally restructure the soil fungal community in a temperate forest. fungal Ecol 2016; 23: 48-57
  72. 72. Corrales A, Turner BL, Tedersoo L, et al. Nitrogen addition alters ectomycorrhizal fungal communities and soil enzyme activities in a tropical montane forest. fungal Ecol 2017; 27: 14-23
  73. 73. Zhu H, Dancik BP, Higginbotham KO. Regulation of extracellular proteinase production in an ectomycorrhizal fungus Hebeloma crustuliniforme. Mycologia 1994; 86: 227-234
  74. 74. Nehls UWE, Kleber R, Wiese J, et al. Isolation and characterization of a general amino acid permease from the ectomycorrhizal fungus Amanita muscaria. New Phytol 1999; 144: 343-349
  75. 75. Javelle A, Morel M, Rodríguez-Pastrana B, et al. Molecular characterization, function and regulation of ammonium transporters (Amt) and ammonium-metabolizing enzymes (GS, NADP-GDH) in the ectomycorrhizal fungus Hebeloma cylindrosporum. Mol Microbiol 2003; 47: 411-430
  76. 76. Rineau F, Shah F, Smits MM, et al. Carbon availability triggers the decomposition of plant litter and assimilation of nitrogen by an ectomycorrhizal fungus. ISME J 2013; 7: 2010-2022
  77. 77. Casieri L, Lahmidi NA, Doidy J, et al. Biotrophic transportome in mutualistic plant–fungal interactions. Mycorrhiza 2013; 23: 597-625
  78. 78. Nehls U, Plassard C. Nitrogen and phosphate metabolism in ectomycorrhizas. New Phytol 2018; 220: 1047-1058
  79. 79. Becquer A, Guerrero-Galán C, Eibensteiner JL, et al. The ectomycorrhizal contribution to tree nutrition. In: Advances in Botanical Research. Elsevier, 2019, pp. 77-126
  80. 80. Javelle A, Rodrı́guez-Pastrana B-R, Jacob C, et al. Molecular characterization of two ammonium transporters from the ectomycorrhizal fungus Hebeloma cylindrosporum. FEBS Lett 2001; 505: 393-398
  81. 81. Montanini B, Moretto N, Soragni E, et al. A high-affinity ammonium transporter from the mycorrhizal ascomycete Tuber borchii. Fungal Genet Biol 2002; 36: 22-34
  82. 82. Willmann A, Weiß M, Nehls U. Ectomycorrhiza-mediated repression of the high-affinity ammonium importer gene AmAMT2 in Amanita muscaria. Curr Genet 2007; 51: 71
  83. 83. Kemppainen MJ, Pardo AG. pHg/pSILBAγ vector system for efficient gene silencing in homobasidiomycetes: optimization of ihpRNA–triggering in the mycorrhizal fungus Laccaria bicolor. Microb Biotechnol 2010; 3: 178-200
  84. 84. Jargeat P, Rekangalt D, Verner M-C, et al. Characterisation and expression analysis of a nitrate transporter and nitrite reductase genes, two members of a gene cluster for nitrate assimilation from the symbiotic basidiomycete Hebeloma cylindrosporum. Curr Genet 2003; 43: 199-205
  85. 85. Kohler A, Kuo A, Nagy LG, et al. Convergent losses of decay mechanisms and rapid turnover of symbiosis genes in mycorrhizal mutualists. Nat Genet 2015; 47: 410-415
  86. 86. Shah F, Nicolás C, Bentzer J, et al. Ectomycorrhizal fungi decompose soil organic matter using oxidative mechanisms adapted from saprotrophic ancestors. New Phytol 2016; 209: 1705-1719
  87. 87. Lindahl BD, Taylor AFS. Occurrence of N-acetylhexosaminidase-encoding genes in ectomycorrhizal basidiomycetes. New Phytol 2004; 164: 193-199
  88. 88. Miyauchi S, Kiss E, Kuo A, et al. Large-scale genome sequencing of mycorrhizal fungi provides insights into the early evolution of symbiotic traits. Nat Commun 2020; 11: 1-17
  89. 89. Talbot JM, Treseder KK. Controls over mycorrhizal uptake of organic nitrogen. Pedobiologia (Jena) 2010; 53: 169-179
  90. 90. Näsholm T, Högberg P, Franklin O, et al. Are ectomycorrhizal fungi alleviating or aggravating nitrogen limitation of tree growth in boreal forests? New Phytol 2013; 198: 214-221
  91. 91. Nicolás C, Martin-Bertelsen T, Floudas D, et al. The soil organic matter decomposition mechanisms in ectomycorrhizal fungi are tuned for liberating soil organic nitrogen. ISME J 2019; 13: 977-988
  92. 92. Lindahl BD, Ihrmark K, Boberg J, et al. Spatial separation of litter decomposition and mycorrhizal nitrogen uptake in a boreal forest. New Phytol 2007; 173: 611-620
  93. 93. Talbot JM, Allison SD, Treseder KK. Decomposers in disguise: mycorrhizal fungi as regulators of soil C dynamics in ecosystems under global change. Funct Ecol 2008; 22: 955-963
  94. 94. Sinsabaugh RL. Phenol oxidase, peroxidase and organic matter dynamics of soil. Soil Biol Biochem 2010; 42: 391-404
  95. 95. Hall IR, Zambonelli A. Laying the foundations. In: Edible ectomycorrhizal mushrooms. Springer, 2012, pp. 3-16
  96. 96. Poitou N, Mamoun M, Ducamp M, et al. After Boletus granulatus, Lactarius deliciosus fructification is obtained in the field from inoculated plants. PHM Rev Hortic 1984; 244: 65-68
  97. 97. Guerin-Laguette A, Cummings N, Butler RC, et al. Lactarius deliciosus and Pinus radiata in New Zealand: towards the development of innovative gourmet mushroom orchards. Mycorrhiza 2014; 24: 511-523
  98. 98. Wang R, Guerin-Laguette A, Yu F-Q. Optimum media for hyphal growth and mycorrhizal synthesis of two Lactarius species. Mycosystema 2020; 39: 1346-1355
  99. 99. Tan ZM, Danell E, Shen AR, et al. Successful cultivation of Lactarius hatsutake—an evaluation with molecular methods. Acta Edulis Fungi 2008; 15: 85-88
  100. 100. Visnovsky SB, Guerin-Laguette A, Wang Y, et al. Traceability of marketable Japanese shoro in New Zealand: using multiplex PCR to exploit phylogeographic variation among taxa in the Rhizopogon subgenus Roseoli. Appl Environ Microbiol 2010; 76: 294-302
  101. 101. Yamanaka K. Commercial cultivation of Lyophyllum shimeji. GAMU GmbH, Institut für Pilzforschung, 2008; pp. 197-202
  102. 102. Hobbie EA, Högberg P. Nitrogen isotopes link mycorrhizal fungi and plants to nitrogen dynamics. New Phytol 2012; 196: 367-382
  103. 103. Daza A, Manjón JL, Camacho M, et al. Effect of carbon and nitrogen sources, pH and temperature on in vitro culture of several isolates of Amanita caesarea (Scop.: Fr.) Pers. Mycorrhiza 2006; 16: 133-136
  104. 104. Tedersoo L, Naadel T, Bahram M, et al. Enzymatic activities and stable isotope patterns of ectomycorrhizal fungi in relation to phylogeny and exploration types in an afrotropical rain forest. New Phytol 2012; 195: 832-843
  105. 105. Endo N, Gisusi S, Fukuda M, et al. In vitro mycorrhization and acclimatization of Amanita caesareoides and its relatives on Pinus densiflora. Mycorrhiza 2013; 23: 303-315
  106. 106. Yamanaka K, Namba K, Tajiri A. Fruit body formation ofBoletus reticulatus in pure culture. Mycoscience 2000; 41: 189
  107. 107. Ohta A, Fujiwara N. Fruit-body production of an ectomycorrhizal fungus in genus Boletus in pure culture. Mycoscience 2003; 44: 295-300
  108. 108. Ohta A. Fruit-body production of two ectomycorrhizal fungi in the genus Hebeloma in pure culture. Mycoscience 1998; 39: 15-19
  109. 109. Peksen A, Kibar B, Yakupoglu G. Favourable culture conditions for mycelial growth of Hydnum repandum, a medicinal mushroom. African J Tradit Complement Altern Med 2013; 10: 431-434
  110. 110. Davis EE, Jong SC. Basidiocarp formation by Laccaria laccata in agar culture. Mycologia 1976; 68: 211-214
  111. 111. Taylor AFS, Högbom L, Högberg M, et al. Natural 15N abundance in fruit bodies of ectomycorrhizal fungi from boreal forests. New Phytol 1997; 136: 713-720
  112. 112. Itoo ZA, Reshi ZA. Effect of different nitrogen and carbon sources and concentrations on the mycelial growth of ectomycorrhizal fungi under in-vitro conditions. Scand J For Res 2014; 29: 619-628
  113. 113. Finlay RD, Frostegård Å, Sonnerfeldt A. Utilization of organic and inorganic nitrogen sources by ectomycorrhizal fungi in pure culture and in symbiosis with Pinus contorta Dougl. ex Loud. New Phytol 1992; 120: 105-115
  114. 114. Yamanaka T. Utilization of inorganic and organic nitrogen in pure cultures by saprotrophic and ectomycorrhizal fungi producing sporophores on urea-treated forest floor. Mycol Res 1999; 103: 811-816
  115. 115. Ohta A. Production of fruit-bodies of a mycorrhizal fungus, Lyophyllum shimeji, in pure culture. Mycoscience 1994; 35: 147-151
  116. 116. Sun Q, Li J, Finlay RD, et al. Oxalotrophic bacterial assemblages in the ectomycorrhizosphere of forest trees and their effects on oxalate degradation and carbon fixation potential Oxalotrophic bacterial assemblages in the ectomycorrhizosphere of forest trees and their effects on oxala. Chem Geol 2019; 514: 54-64
  117. 117. Lazarević J, Stojičić D, Keča N. Effects of temperature, pH and carbon and nitrogen sources on growth of in vitro cultures of ectomycorrhizal isolates from Pinus heldreichii forest. For Syst 2016; 25: 3
  118. 118. Terashima Y. Carbon and nitrogen utilization and acid production by mycelia of the ectomycorrhizal fungusTricholoma bakamatsutake in vitro. Mycoscience 1999; 40: 51
  119. 119. Hamada M. Physiology and ecology of Armillaria matsutake. Bot Mag 1950; 63: 40-41
  120. 120. Vaario LM, Sah SP, Norisada M, et al. Tricholoma matsutake may take more nitrogen in the organic form than other ectomycorrhizal fungi for its sporocarp development: the isotopic evidence. Mycorrhiza 2019; 29: 51-59
  121. 121. Kawai M, Abe S. Studies on the artificial reproduction of Tricholoma matsutake (S. ito et Imai) Sing. i. effects of carbon and nitrogen sources in media on the vegetative growth of Tricholoma matsutake. Transactions. 1976
  122. 122. Yamada A, Maeda K, Kobayashi H, et al. Ectomycorrhizal symbiosis in vitro between Tricholoma matsutake and Pinus densiflora seedlings that resembles naturally occurring ‘shiro’. Mycorrhiza 2006; 16: 111-116
  123. 123. Kibar B, Peksen A. Nutritional and environmental requirements for vegetative growth of edible ectomycorrhizal mushroom Tricholoma terreum. Agriculture 2011; 98: 409-414
  124. 124. Liu R-S, Li D-S, Li H-M, et al. Response surface modeling the significance of nitrogen source on the cell growth and Tuber polysaccharides production by submerged cultivation of Chinese truffle Tuber sinense. Process Biochem 2008; 43: 868-876
  125. 125. Bonet JA, Oliach D, Fischer C, et al. Cultivation methods of the black truffle, the most profitable mediterranean non-wood forest product; a state of the art review. In EFI proceedings 2009; 57: 57-71
  126. 126. Guerin-Laguette A, Plassard C, Mousain D. Effects of experimental conditions on mycorrhizal relationships between Pinus sylvestris and Lactarius deliciosus and unprecedented fruit-body formation of the Saffron milk cap under controlled soilless conditions. Can J Microbiol 2000; 46: 790-799
  127. 127. Parladé J, Pera J, Luque J. Evaluation of mycelial inocula of edible Lactarius species for the production of Pinus pinaster and P. sylvestris mycorrhizal seedlings under greenhouse conditions. Mycorrhiza 2004; 14: 171-175
  128. 128. Águeda B, Parladé J, Fernández-Toirán LM, et al. Mycorrhizal synthesis between Boletus edulis species complex and rockroses (Cistus sp.). Mycorrhiza 2008; 18: 443-449
  129. 129. Oria-de-Rueda JA, Hernández-Rodríguez M, Martín-Pinto P, et al. Could artificial reforestations provide as much production and diversity of fungal species as natural forest stands in marginal Mediterranean areas? For Ecol Manage 2010; 260: 171-180
  130. 130. Yamanaka T, Yamada A, Furukawa H. Advances in the cultivation of the highly-prized ectomycorrhizal mushroom Tricholoma matsutake. Mycoscience 2020; 61: 49-57
  131. 131. Ogawa M. Microbial ecology of mycorrhizal fungus, Tricholoma matsutake Sing. in pine forest 1. Fungal colony (‘ Shiro’) of T. matsutake. Bull Prod Res Inst 1975; 272: 79-121
  132. 132. Narimatsu M, Koiwa T, Sakamoto Y, et al. Estimation of novel colony establishment and persistence of the ectomycorrhizal basidiomycete Tricholoma matsutake in a Pinus densiflora forest. fungal Ecol 2016; 24: 35-43
  133. 133. Hortal S, Plett KL, Plett JM, et al. Role of plant–fungal nutrient trading and host control in determining the competitive success of ectomycorrhizal fungi. ISME J 2017; 11: 2666-2676
  134. 134. Terashita T, Kono M. Carboxyl proteinases from Tricholoma matsutake and its related species. Mem Fac Agric Kinki Univ 1989; 22:5-12
  135. 135. Terashita T, Kono M, Yoshikawa K, et al. Productivity of hydrolytic enzymes by mycorrhizal mushrooms. Mycoscience 1995; 36: 221-225
  136. 136. Kusuda M, Ueda M, Konishi Y, et al. Detection of β-glucosidase as saprotrophic ability from an ectomycorrhizal mushroom, Tricholoma matsutake. Mycoscience 2006; 47: 184-189
  137. 137. Vaario L-M, Guerin-Laguette A, Matsushita N, et al. Saprobic potential of Tricholoma matsutake: growth over pine bark treated with surfactants. Mycorrhiza 2002; 12: 1-5
  138. 138. Vaario L-M, Heinonsalo J, Spetz P, et al. The ectomycorrhizal fungus Tricholoma matsutake is a facultative saprotroph in vitro. Mycorrhiza 2012; 22: 409-418
  139. 139. Endo N, Dokmai P, Suwannasai N, et al. Ectomycorrhization of Tricholoma matsutake with Abies veitchii and Tsuga diversifolia in the subalpine forests of Japan. Mycoscience 2015; 56: 402-412
  140. 140. Yamada A, Ogura T, Degawa Y, et al. Isolation of Tricholoma matsutake and T. bakamatsutake cultures from field-collected ectomycorrhizas. Mycoscience 2001; 42: 43-50
  141. 141. Van Der Eerden L, De Vries W, Van Dobben H. Effects of ammonia deposition on forests in the Netherlands. Atmos Environ 1998; 32: 525-532
  142. 142. Nohrstedt HO. Fruit-body production and 137Cs-activity of Cantharellus cibarius after nitrogen and potassium fertilization. Forestry Research Inst. of Sweden, 1994
  143. 143. Harley JL, Smith SE. Mycorrhizal Symbiosis Academic Press. London/New York. 1983
  144. 144. Richards BN. Mycorrhiza development of loblolly pine seedlings in relation to soil reaction and the supply of nitrate. Plant Soil 1965; 22: 187-199
  145. 145. Takeshi I, Ogawa M. Cultivating method of the mycorrhizal fungus, Tricholoma matsutake (Ito et Imai) Sing. (II) increasing number of Shiro (fungal colony) of T. matsutake by thinning the understory vegetation. J Jpn For Soc 1979; 61: 163-173
  146. 146. Baar J, Ter Braak CJF. Ectomycorrhizal sporocarp occurrence as affected by manipulation of litter and humus layers in Scots pine stands of different age. Appl Soil Ecol 1996; 4: 61-73
  147. 147. Gundersen P, Callesen I, De Vries W. Nitrate leaching in forest ecosystems is related to forest floor CN ratios. Environ Pollut 1998; 102: 403-407
  148. 148. Vaario LM, Kiikkilä O, Hamberg L. The influences of litter cover and understorey vegetation on fruitbody formation of Tricholoma matsutake in southern Finland. Appl Soil Ecol 2013; 66: 56-60
  149. 149. Yun W, Hall IR. Edible ectomycorrhizal mushrooms: challenges and achievements. Can J Bot 2004; 82: 1063-1073
  150. 150. Smit E, Veenman C, Baar J. Molecular analysis of ectomycorrhizal basidiomycete communities in a Pinus sylvestris L. stand reveals long-term increased diversity after removal of litter and humus layers. FEMS Microbiol Ecol 2003; 45: 49-57
  151. 151. Bogar L, Peay K, Kornfeld A, et al. Plant-mediated partner discrimination in ectomycorrhizal mutualisms. Mycorrhiza 2019; 29: 97-111
  152. 152. Garbaye J. Tansley review no. 76 helper bacteria: a new dimension to the mycorrhizal symbiosis. New Phytol 1994; 128: 197-210
  153. 153. Paul LR, Chapman BK, Chanway CP. Nitrogen fixation associated with Suillus tomentosus tuberculate ectomycorrhizae on Pinus contorta var. latifolia. Ann Bot 2007; 99: 1101-1109
  154. 154. Barbieri E, Ceccaroli P, Saltarelli R, et al. New evidence for nitrogen fixation within the Italian white truffle Tuber magnatum. Fungal Biol 2010; 114: 936-942
  155. 155. Danell E, Alström S, Ternström A. Pseudomonas fluorescens in association with fruit bodies of the ectomycorrhizal mushroom Cantharellus cibarius. Mycol Res 1993; 97: 1148-1152
  156. 156. Oh S-Y, Lim YW. Root-associated bacteria influencing mycelial growth of Tricholoma matsutake (pine mushroom). J Microbiol 2018; 56: 399-407
  157. 157. Vaario L-M, Asamizu S, Sarjala T, et al. Bioactive properties of streptomyces may affect the dominance of Tricholoma matsutake in shiro. Symbiosis 2020; 81: 1-13
  158. 158. Oh S-Y, Kim M, Eimes JA, et al. Effect of fruiting body bacteria on the growth of Tricholoma matsutake and its related molds. PLoS One 2018; 13: e0190948

Written By

Lu-Min Vaario and Norihisa Matsushita

Submitted: 20 September 2020 Reviewed: 07 December 2020 Published: 29 January 2021