Janice Pease (315)328-5793 Janice.Pease@Yahoo.Com 130 Beebe Rd, Potsdam, N.Y

Janice Pease (315)328-5793 [email protected] 130 Beebe Rd, Potsdam, N.Y. 13676

June 18, 2018

Via Email

Honorable Kathleen H. Burgess, Secretary to the PSC Re: Case 16- F-0268, Application of Atlantic Wind LLC for a certiﬁcate of Environmental Compatibility and Public Need Pursuant to Article 10 for Construction of the North Ridge Wind Energy Project in the Towns of Parishville and Hopkinton, St. Lawrence County.

Dear Secretary Burgess: The North Ridge Wind Facility is a threat to our local ecosystems and will decrease our biodiversity. The forests, wetlands, and meadows are rich with ﬂora and fauna which are dependent on the relationships between other living organisms, at a microscopic level. While the relationship between an organism and its environment may not be visible to the naked eye, they may still make enormous contributions to the health of our local ecosystems. Fungi are wonderful contributors despite the lack of acknowledgment. Most are familiar with mushrooms such as crimini, button, portabella, and shitake, however other fungi which play a larger role in the health of our environment. While hiking through Hopkinton (both North and South of Route 72), I have seen many varieties of mushrooms which I was unfamiliar with and realized I needed to pay closer attention to.

I am certain that the environmental agencies and ofﬁcials determining the environmental impact of this project are not considering the role of fungi, nor are they looking for ways to mitigate the impact this project would have on fungi.

I can say, without pause, that fungi are as important to an ecosystem as any other component.

Fungi/mushrooms are made up of thin thread-like mycelium called hyphae, which create a mycelium network. This network connects to many plants/trees, linking through the hyphae, redistributing nutrients through the aquatic and terrestrial communities which make up an ecosystem. The fungi absorb nutrients through the mycelium which secrete enzymes, either onto or into plant matter, breaking down this material into a usable form for food. Their role in decomposition makes mycelium intrinsically vital to the health of ecosystems. Biodiversity creates balance and that balance keeps an ecosystem healthy and able to ﬁght off pathogens/pests.

Networks of mycelium can be enormous, the largest known is in Oregon.

“More precisely, a speciﬁc honey fungus measuring 2.4 miles (3.8 km) across in the Blue Mountains in Oregon is thought to be the largest living organism on Earth.” http://www.bbc.com/earth/story/20141114-the- biggest-organism-in-the-world

“The discovery of this giant Armillaria ostoyae in 1998 heralded a new record holder for the title of the world's largest known organism, believed by most to be the 110-foot- (33.5-meter-) long, 200-ton blue whale. Based on its current growth rate, the fungus is estimated to be 2,400 years old but could be as ancient as 8,650 years, which would earn it a place among the oldest living organisms as well.” https://www.scientiﬁcamerican.com/article/strange-but-true-largest- organism-is-fungus/

“This one, A. ostoyae, causes Armillaria root disease, which kills swaths of conifers in many parts of the U.S. and Canada. The fungus primarily grows along tree roots via hyphae, fine filaments that mat together and excrete digestive enzymes. But Armillaria has the unique ability to extend rhizomorphs, flat shoestringlike structures, that bridge gaps between food sources and expand the fungus's sweeping perimeter ever more.” https://www.scientificamerican.com/article/strange-but-true-largest- organism-is-fungus/

“A combination of good genes and a stable environment has allowed this particularly ginormous fungus to continue its creeping existence over the past millennia. "These are very strange organisms to our anthropocentric way of thinking," says biochemist Myron Smith of Carleton University in Ottawa, Ontario. An Armillaria individual consists of a network of hyphae, he explains. "Collectively, this network is called the mycelium and is of an indeﬁnite shape and size.” https:// www.scientiﬁcamerican.com/article/strange-but-true-largest-organism-is- fungus/

“However, the pathogenic species only infect their hosts under environmental conditions that favor development of the fungus or that weakens the host resistance. Conditions allowing abundant development of soil inoculum of Armillaria make hosts more prone to infection, while at constant inoculum density infection is more likely to occur when trees are weakened. Armillaria species can survive for long periods in colonized wood or as rhizomorphs. The opportunistic life style of Armillaria is explained by its ability to persist when there are no food sources, waiting for conditions that allow the weakening of the host. “ https://projects.ncsu.edu/cals/course/pp728/Armillaria/Armillaria.htm

The effects of certain species of mycelium can be the determining factor in a forests decline. By weakening the ecosystem through industrialization and increased pollution the trees lose their resilience and become prone to infestation from pathogens/pests.

Mycelial mats growing in different landscapes have unique fungi species, creating an individualized base for abundantly different growth. Even within the same ecosystem, different species of fungi create distinct ecological communities (microbiomes) with the potential to support vastly different microorganisms (organisms typically consisting of 1 cell- fungi, algae, bacteria, protozoa), ﬂora (plant), and fauna (animal) species. The mycelia support this individual balance, creating the base of that particular ecosystem through the plants which feed the animals. The mycelial landscape networks are the foundation of the aquatic and terrestrial food webs, shaping the ecosystem from the ground up… literally.

Natural ecosystems are important and difﬁcult to replicate, there is a balance that is achieved through the process which evolves slowly over many years. This entire planet is dependent on the individual ecosystems and their health extending out.

The connection of communities/ecosystems is not well understood, the role of mycelium is being researched. Myselium can colonize a forest ﬂoor, creating a symbiotic relationship which aids in moisture retention, erosion prevention, nutrient absorption, and provides resilience to pathogens.

Mycelium create this symbiotic relationship with host trees, as mycelium cannot photosynthesize they are dependent on other organisms for their sustenance. By creating interconnected networks between the tree roots the fungi are able to exchange of nutrients. Different fungi have different methods for creating these interconnected colonies. Some soil-dwelling fungi (Arbuscular mycorrhizas) penetrate the host, while others surround the host root without penetrating (Ectomycorrhizas). By breaking down and transforming organic decaying matter (leaves, bark, etc.) into soil, the mycelium feed a forest, giving life to trees and plants. Fungi is giving life while taking life simultaneously. This act inadvertently feeds animals which is continues through the food chain, we are all dependent on mycelium. The importance of mycelium is understated, this silent contribution shapes an ecosystem, impacting the health of the unique and diverse biological communities (biomes).

With every lost potential host plant/tree we lose more environmental integrity. By replacing forests, wetlands, and meadows with gravel, concrete, asphalt, etc. we are creating imbalance which is echoed world wide.

Despite the opinions of “experts” who are weighing in on these land use policies which are impacting local communities, I have yet to hear any discussion of the impact these giant industrial factories have on climate change. Speciﬁc attention needs to be paid to the relationship between deforestation creating the loss of precious carbon sinks. More research is needed concerning the transference of carbon from one plant to another and the role of individual species in reducing carbon in the atmosphere (https://onlinelibrary.wiley.com/doi/full/10.1111/j. 1461-0248.2004.00574.x). The wider ecological impact of improperly sited industrial facilities is not well understood.

What we do not know far outweighs what we do know.

Respectfully, Janice Pease Janice Pease

*electronically signed

Attached:

*Ectomycorrhizal fungi: exploring the mycelial frontier Ian C. Anderson1 & John W.G. Cairney2

*What Are Mycelia in Microbiology? By Andrea Becker; Updated April 25, 2017 Ectomycorrhizal fungi: exploring the mycelial frontier Ian C. Anderson1 & John W.G. Cairney2

1The Macaulay Institute, Craigiebuckler, Aberdeen, UK; and 2Centre for Plant and Food Science, University of Western Sydney, NSW, Australia

Correspondence: Ian C. Anderson, The Abstract Macaulay Institute, Craigiebuckler, Aberdeen AB15 8QH, UK. Tel.: 144 1224 498200; Ectomycorrhizal (ECM) fungi form mutualistic symbioses with many tree species ext. 2357; fax: 144 1224 498207; e-mail: and are regarded as key organisms in nutrient and carbon cycles in forest [email protected] ecosystems. Our appreciation of their roles in these processes is hampered by a lack of understanding of their soil-borne mycelial systems. These mycelia represent Received 1 December 2006; revised 12 the vegetative thalli of ECM fungi that link carbon-yielding tree roots with soil February 2007; accepted 5 March 2007. nutrients, yet we remain largely ignorant of their distribution, dynamics and First published online 28 April 2007. activities in forest soils. In this review we consider information derived from investigations of fruiting bodies, ECM root tips and laboratory-based microcosm DOI:10.1111/j.1574-6976.2007.00073.x studies, and conclude that these provide only limited insights into soil-borne ECM mycelial communities. Recent advances in understanding soil-borne mycelia of Editor: Ramon Diaz Orejas ECM fungi have arisen from the combined use of molecular technologies and

Keywords novel ﬁeld experimentation. These approaches have the potential to provide ectomycorrhizal fungi; soil-borne mycelia; unprecedented insights into the functioning of ECM mycelia at the ecosystem mycelial biomass; ectomycorrhizal level, particularly in the context of land-use changes and global climate change.

communities; elevated atmospheric CO2.

root is functionally critical to the mutualism, as it represents Introduction the interface across which nutrients and carbon are trans- Many tree species in forest habitats worldwide rely upon ferred between the partners (Smith & Read, 1997). mutualistic ectomycorrhizal (ECM) fungi to fulfil their In addition to the structures formed at the host root, nutrient requirements (Smith & Read, 1997). These fungi ECM fungi produce mycelia that extend from the mantle contribute to tree nutrition by means of mineral weathering into the surrounding soil (Fig. 1), although the extent and (Landeweert et al., 2001) and mobilization of nutrients from structure of this extramatrical mycelium is thought to differ organic complexes (Read & Perez-Moreno, 2003). They are between ECM fungal taxa (Agerer, 2001). The soil-borne also an important avenue for the delivery of carbon to soil mycelia of ECM fungi are regarded as functionally impor- and are responsible for a substantial component of forest- tant to the mutualism in foraging for, and translocation of, soil carbon fluxes (Soderstr¨ om,¨ 1992; Hogberg¨ et al., 2001; nutrients and water. They also infect short lateral roots Hogberg¨ & Hogberg,¨ 2002; Godbold et al., 2006; Hobbie, (Smith & Read, 1997) and potentially form a common 2006). ECM fungi are thus regarded as key elements of forest mycelial network that might facilitate carbon and/or nutri- nutrient cycles and as strong drivers of forest ecosystem ent movement between individual tree hosts (Simard & processes (Read et al., 2004). Durall, 2004; Selosse et al., 2006). At the ecosystem level The fungi that form ECM associations comprise a tax- these mycelia are of further significance in the mobilization onomically broad suite of basidiomycetes and, to a lesser of nutrients from organic and recalcitrant inorganic sources, extent, ascomycetes (Smith & Read, 1997). Typically, the both in competition and in cooperation with soil-dwelling fungi form a mycelial mantle around short lateral roots of saprotrophs, in the relocation of nutrients within the their hosts and penetrate between epidermal and cortical soil–plant continuum, and in the delivery and distribution cells, surrounding them with a highly branched structure, of carbon belowground (Leake et al., 2002; Wallander, the Hartig net (Peterson et al., 2004). Significant variation 2006). Despite the importance of soil-borne mycelia of exists in the morphology of ECM root tips that are infected ECM fungi in forest ecosystems, along with their likely by different fungal taxa, and analysis of macroscopic and importance in the global carbon budget (Hobbie, 2006) microscopic characteristics of ECM roots is used widely for and sensitivity to global change factors such as rising atmo-

identiﬁcation of the ECM fungi (Agerer, 1987–2002). The spheric CO2 (Rillig et al., 2002), we know relatively little fungus–plant interface formed by the Hartig net in the ECM regarding their extent, diversity and spatial location in forest

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soils. In particular, the current lack of fundamental informa- no insight into the extent of their mycelial systems in soil tion regarding the physical and physiological continuity of (Genney et al., 2006), nor into their contributions to belowground mycelia limits our understanding of the im- nutrient and carbon cycling. portance of ECM mycelial networks in nutrient cycling and To date, investigation of ECM mycelia in soil has been in the distribution of carbon and nutrients within forest-soil constrained by the difﬁculty in identifying ECM mycelia and systems (Cairney, 2005). Furthermore, although many de- in separating these from mycelia of non-ECM soil fungi. tailed investigations of communities of ECM fungi coloniz- Most of our current understanding has thus been derived via ing root tips have been undertaken (see below), the relative inference from the distribution of fruiting bodies and ECM importance of particular taxa in root-tip communities gives root tips in the ﬁeld and/or from observations of mycelia

Fig. 1. Mycelial systems of ectomycorrhizal fungi. (a) A typical microcosm system demonstrating the mycelium of Suillus variegatus growing from a Scots pine seedling (photo taken by P.M.A. Fransson). (b) Russula sp. fruiting body and associated mycelium. (c) A close-up of (b) showing the base of the fruiting-body stipe, the associated white mycelium, and ECM root tips (arrows) colonized by this fungus. (d) A dense mycelial mat that has been lifted from a rock surface in a native Scots pine forest. Patches of brown, pink, white and yellow mycelium of different fungal species are visible (photo taken by C.D. Campbell).

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produced in microcosms in the laboratory (Fig. 1). As we ECM fungi are difficult or impossible to isolate into axenic will outline in this review, many of the assumptions upon culture by conventional methods (Brundrett et al., 1996), which these inferences are based are somewhat tenuous, and limiting the usefulness of somatic compatibility testing for current understanding of complex ECM mycelial systems in ECM fungi. Concerns have also been raised about the forest soils remains limited. Recent years have, however, resolution of the method for identifying genotypes of some witnessed the development and application of a suite of ECM taxa (Sen, 1990; Jacobson et al., 1993). molecular methods that are yielding unprecedented insights More recently, molecular methods for the identification into ECM mycelia in forest soils. In this review we highlight of genotypes using DNA extracted from vegetative fruiting- some of the limitations of our current knowledge, along body tissue have been used in this context. A range of with the most significant recent advances in our apprecia- dominant molecular markers, including random amplified tion of the distribution, dynamics and activities of soil- polymorphic DNA (RAPD), simple sequence repeat (SSR), borne ECM mycelial systems along with their responses to amplified fragment length polymorphism (AFLP), single-

elevated atmospheric CO2 concentrations. strand conformational polymorphism (SSCP) and inter- retrotransposon amplified polymorphism (IRAP) markers Fruiting bodies as indicators of (e.g. De la Bastide et al., 1994; Anderson et al., 1998; Bonello community structure et al., 1998; Sawyer et al., 1999; Redecker et al., 2001; Murata et al., 2005), along with codominant repeat SSR markers Although frequently used in surveys of higher fungal (e.g. Zhou et al., 2001a; Dunham et al., 2003; Kretzer et al., diversity, fruiting bodies provide little useful information 2004; Wu et al., 2005; Bergemann et al., 2006), has now been regarding the diversity of ECM fungal mycelia in soil. used to infer the genotype distributions of certain ECM Clearly, the presence of an epigeous fruiting body indicates fungi in a variety of forest habitats. While some of these that mycelium of the fungus must be present in one form or molecular methods may not resolve all genotypes in a another in the underlying soil. Because many ECM taxa population (Redecker et al., 2001; Dunham et al., 2003; produce no epigeous fruiting bodies, or may not have Bagley & Orlovich, 2004), they have been widely used to fruited during the duration of the survey, it is impossible to estimate the spatial distribution of ECM fungal genotypes. infer the importance of a fungus in the belowground It is thus evident that genotypes of some ECM fungi are community on the basis of fruiting-body observations. In spread over tens of metres in some forests, implying that fact, where fruiting-body data have been compared with mycelia can extend for considerable distances, and persist ECM root tips (see below), there is generally a very poor for several years, in the field (e.g. Dahlberg & Stenlid, 1990; correlation between the two, with the latter typically indi- Baar et al., 1994; Dahlberg, 1997; Anderson et al., 1998; cating greater species richness (Erland & Taylor, 2002). Bonello et al., 1998; Sawyer et al., 1999). Estimates of Furthermore, there is evidence that, for Laccaria spp. at belowground mycelial growth rates (e.g. Dahlberg & Stenlid, least, physiological and/or genetic differences may influence 1990; Bonello et al., 1998; Sawyer et al., 1999; Lian et al., fruiting at the intraspecific level (Selosse et al., 2001). 2006) and investigations of the survival of genotypes introduced as ECM inoculum in forest plantations (e.g. De la Estimation of mycelial distribution using Bastide et al., 1994; Selosse et al., 1998; Sawyer et al., 2001) fruiting bodies provide strong supplementary evidence that mycelia of certain ECM fungi can persist in forest soils for at least tens For those ECM fungi that produce epigeous fruiting bodies, of years. Whether these genotypes persist in soil as contin- their presence has been used to infer the distribution of uous mycelia [ = genets (Dahlberg & Stenlid, 1995)], or have mycelia of the fungi in forest soils. This method centres on fragmented into smaller mycelia [ = ramets (Dahlberg & analysis of the mycelial genotype in the vegetative tissue of Stenlid, 1995)] as a result of, for example, feeding activities the fruiting body and mapping the distribution of the of mycophagous soil arthropods (Schneider et al., 2005) or genotypes in a forest stand. Fruiting bodies that have the other forms of disturbance, remains unclear. same vegetative genotype must have developed from a Fruiting-body collections indicate that multiple geno- genetically identical mycelium that has grown through soil, types of some species, such as Laccaria spp., frequently thus allowing estimation of the distribution of soil-borne occur in an area of a few square metres, suggesting the mycelia (Dahlberg & Stenlid, 1995). Initially this was presence of many spatially restricted and ephemeral mycelia achieved by isolation of vegetative fruiting-body stipe my- in the underlying soil (e.g. Gryta et al., 1997; Gherbi et al., celium into axenic culture, followed by somatic incompat- 1999; Fiore-Donno & Martin, 2001; Dunham et al., 2003; ibility testing to identify which isolates are genetically Murata et al., 2005). Within genera such as Amanita identical (e.g. Dahlberg & Stenlid, 1990, 1994; Baar et al., (Redecker et al., 2001; Sawyer et al., 2001, 2003; Bagley & 1994). While useful for fungi that are readily cultured, many Orlovich, 2004; Liang et al., 2005), Russula (Redecker et al.,

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2001; Bergemann & Miller, 2002; Liang et al., 2004; Riviere gations in recent years (Horton & Bruns, 2001; Anderson, et al., 2006) or Tricholoma (Murata et al., 2005; Gryta et al., 2006), and a great deal of thought has been invested in 2006; Lian et al., 2006) there are varying reports of wide- designing appropriate sampling strategies (e.g. Horton & spread and spatially restricted genotypes. Similar observa- Bruns, 2001; Taylor, 2002). Although some specific habitats tions have been made in population studies of individual appear to be characterized by low species diversity of ECM species such as Suillus bovinus (Dahlberg & Stenlid, 1990, roots (Chambers et al., 2005), numerous such investigations 1994), Laccaria bicolor (Baar et al., 1994; Selosse et al., 1998) have revealed that communities of ECM roots in multi- and Pisolithus albus (Anderson et al., 1998, 2001), making it farious forest habitats are generally extremely species-rich difficult to predict genotype distribution, and so infer the (reviewed Horton & Bruns, 2001). It is also evident that dimensions of belowground mycelia, strictly on the basis of communities of ECM roots vary with vegetation/environ- taxonomic affiliation. mental gradients and chronosequences (e.g. Kernaghan & The occurrence of multiple spatially restricted genotypes Harper, 2001; Wurzenburger et al., 2004; Dickie & Reich, of an ECM fungus is thought to reflect frequent mycelial 2005; Palfner et al., 2005; Robertson et al., 2006). establishment from meiospores, while a local population ECM root species richness and/or community structure dominated by widespread genotypes is characteristic of the are influenced by a range of factors, including soil type sustained growth of persistent belowground mycelia (Dahl- (Gehring et al., 1998; Moser et al., 2005), soil moisture (e.g. berg & Stenlid, 1995). There is some evidence that, for Taylor & Alexander, 1989; Kar˚ en´ et al., 1996; Shi et al., 2002; certain ECM fungi, the former may prevail in recently Walker et al., 2005), season (Giachini et al., 2004), natural disturbed or younger forests, while the latter predominate nutrient gradients (Toljander et al., 2006), fire (e.g. Visser, in mature undisturbed forests (Dahlberg & Stenlid, 1990, 1995; Stendell et al., 1999; Grogan et al., 2000; Smith et al., 1995). This is not always the case, however, because the 2005; and see the review by Cairney & Bastias, 2006), distribution of host tree species in a mixed forest may activities of herbivores and plant parasites (Brown et al., influence the distribution of ECM genotypes (Zhou et al., 2001; Cullings et al., 2005; Mueller & Gehring, 2006), and 2000). Furthermore, multiple spatially restricted genotypes the quality and quantity of organic matter (e.g. Conn & have been identified in populations of some ECM fungi in Dighton, 2000; Cullings et al., 2003). ECM root-tip com- mature forests (e.g. Gherbi et al., 1999; Fiore-Donno & munities can also be strongly influenced by a range of forest Martin, 2001; Redecker et al., 2001), and widespread geno- management practices (reviewed Jones et al., 2003) and by types in relatively young plantations (Selosse, 2003). The gradients of nitrogen deposition (Taylor et al., 2000; Lilles- meaning of these observations in terms of the distribution of kov et al., 2002; Dighton et al., 2004). Further anthropogenic soil-borne mycelia is questionable. At best, such information factors that have been shown to influence ECM root com- based on fruiting-body distribution allows estimation of munities include localized nitrogen addition (e.g. Kar˚ en´ & possible mycelial dimensions in the underlying soil. It Nylund, 1997; Fransson et al., 2000; Peter et al., 2001; Avis provides no information on mycelial continuity, density or et al., 2003; Berch et al., 2006; Carfrae et al., 2006), acid vertical distribution in the soil profile. precipitation (e.g. Rapp & Jentschke, 1994; Qian et al., 1998;

Roth & Fahey, 1998), elevated atmospheric CO2 concentrations (Rey & Jarvis, 1997; Fransson et al., 2001; Kasurinen Community structure and spatial et al., 2005), toxic metal contamination (e.g. Markkola et al., distribution based on ectomycorrhizal 2002), and other forms of anthropogenic pollution (re- root tips viewed Cairney & Meharg, 1999).

Root tip-based assessment of belowground ECM Diversity and distribution of ECM root tips at fungal diversity different scales With the realization that analysis of ECM fungal commu- Although belowground root-tip analyses have been instru- nities on the basis of fruiting-body data is a poor indicator mental in furthering our understanding of ECM fungal of belowground communities of the fungi (Horton & Bruns, communities, it is without doubt that such studies are 2001; Erland & Taylor, 2002) came acceptance that commu- confounded both by sampling intensity and by the scale at nities of ECM fungi are best analysed on the basis of which the study was conducted (Horton & Bruns, 2001; identification and enumeration of short lateral roots in- Taylor, 2002). By constructing species À area curves for data fected by ECM fungal taxa ( = ECM roots). Initially achieved published in four previous studies, Horton & Bruns (2001) by morphological identification of ECM roots (e.g. Taylor & demonstrated that, in all but one, insufficient samples were Alexander, 1989; Massicotte et al., 1999), PCR-based mole- analysed to have covered the diversity of ECM taxa present. cular approaches have become de rigueur for these investi- This demonstrates that a potentially inaccurate or partial

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picture of diversity and community composition can be functionally most important in forest soil nutrient and carbon generated if the sampling intensity is insufficient. While this cycling processes À the soil-borne mycelial systems. alone has consequences for data interpretation, sampling Similar to aboveground and belowground comparisons intensity cannot be considered independently from scale of ECM communities, numerous studies have reported a (Lilleskov et al., 2004). This is particularly important lack of congruence between ECM species colonizing root because in many field studies the desirable scale for the tips and those detected using molecular methods to be analysis is the stand or forest level, but, in reality, pattern in present as mycelia in the same soil volume (Koide et al., most ECM communities occurs at much finer scales. For 2005a; Kjller, 2006). Therefore, the true diversity of ECM example, analysis of ECM root-tip community data from species may remain uncovered if those species that are only eight studies suggested that it is necessary to take cores at present as mycelia in a given soil volume, and which are least 3 m apart in order to achieve the greatest sampling likely to be of some functional importance to their host efficiency for stand-level community analysis (Lilleskov partners, are not considered. In addition, even where intense et al., 2004). However, ECM root-tip abundance and simi- sampling protocols have been adopted, the spatial distribu- larity of community composition have been shown to be tion patterns of root-tip communities may not necessarily highly variable at much finer scales (5–20 cm) (Tedersoo reflect the patterns of the mycelial distribution in soil of the et al., 2003; Izzo et al., 2005), with a complete change in constituent species (Genney et al., 2006). ECM species composition being observed at a scale of 50 cm Because ECM fungi are ‘nonresource-unit-restricted’ in a mixed forest (Tedersoo et al., 2003). fungi (sensu Boddy, 1999), their mycelia forage through soil There is an obvious trade-off between scale and sampling to greater or lesser extents for nutrients, water and new intensity in root tip-based investigations of ECM commu- short-lateral roots to colonize (Read, 1992). In doing so, nities, and alternative sampling approaches, including the they form indeterminate, structurally and physiologically selection of random root tips from soil cores (Horton & heterogeneous interconnected networks of which the colo- Bruns, 2001; Tedersoo et al., 2003), down to as few as three nized roots of the plant host form a part (Rayner, 1991; ECM root-tips per sample (Koide et al., 2005b), have been Cairney & Burke, 1996). There is a growing awareness not suggested as a possible way of tackling this problem. In most only that investigation of ECM mycelia in forest soils is ECM communities, a small number of ECM species are imperative to a functional understanding of ECM commu- highly abundant and dominant (Taylor, 2002). In these nities, but also that communities of ECM fungi when situations it is difficult to see how this would not confound considered from the perspective of their mycelial systems in the ecological interpretation of the data when so few root soil may be quite different from those identified on the basis tips are analysed. of colonized root tips (e.g. Horton & Bruns, 2001; Cairney, 2005; Anderson, 2006; Genney et al., 2006; Wallander, 2006). Vertical stratification in ECM root-tip communities Estimation of mycelial distribution in ectomycorrhizal populations using root A major advance in our understanding of the belowground tips ecology of ECM fungi has been the observation that ECM root-tip communities are vertically stratified in the soil In addition to community analysis, ECM roots have been profile, with different fungi typically occupying different used to estimate the distribution of ECM mycelial genotypes horizons (Goodman & Trofymow, 1998; Rosling et al., 2003; belowground. This work has confirmed the observations Tedersoo et al., 2003; Izzo et al., 2005; Baier et al., 2006; based on fruiting-body distribution (see above) that geno- Genney et al., 2006). While this might signal a degree of types of some taxa are spatially restricted belowground and niche differentiation between ECM taxa, caution is required that the distribution of others can be widespread (Guidot in interpretation because the vertical distribution of ECM et al., 2001; Hirose et al., 2004; Kretzer et al., 2004; Lian roots does not necessarily reflect the distribution of mycelia et al., 2006). It is also clear that genotypes of some ECM of the respective taxa (Genney et al., 2006; and see below). fungal taxa can infect multiple hosts in the field simulta- Despite the extensive and often painstaking effort that has neously (Lian et al., 2006), providing circumstantial support been devoted to investigating communities of ECM roots, our for the notion of a common mycelial network in forest soil understanding of the belowground ecology of ECM fungi in (Peter, 2006). Importantly, it has also become evident that forest ecosystems remains limited. The ECM root-based while estimates of genotype distribution based on fruiting- studies have provided unprecedented insights into the diver- body distribution sometimes correlate roughly with esti- sity of fungal taxa that form ECM associations, along with mates based on root tips (Guidot et al., 2001; Zhou et al., aspects of their dynamics under a range of conditions, yet they 2001b; Lian et al., 2006), in other instances root tip-based have failed to consider the components of ECM fungi that are estimates indicate more widespread genotype distribution

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(Hirose et al., 2004). Furthermore, there is strong evidence Some ECM fungi produce dense mycelial mats or shiro in that belowground genotypes of at least some taxa, can differ forest soil. Mat-forming fungi such as Gaultheria and in temporal fruiting patterns (Guidot et al., 2001; Zhou Hysterangium spp. produce persistent dense mycelia that et al., 2001b; Hirose et al., 2004), meaning that, unless occupy the upper few centimetres in soil and occupy areas repeated sampling is conducted, fruiting-body analysis may that are typically o 1m2 (Cromack et al., 1979; Griffiths only unveil part of the population that is present. Although, et al., 1991). While discrete mats produced by these fungi with an appropriate sampling strategy, root-tip studies may can be identified and measured in soil, it is not clear whether provide some information on the vertical distribution of they comprise single or multiple mycelia of the fungi in mycelial genotypes in ECM roots (Zhou et al., 2001b), they question. The shiro produced by Tricholoma matsutake do not necessarily provide an accurate reflection of the persist for many years and develop by outward growth in a vertical distribution of the equivalent soil-borne mycelia ‘fairy ring’ fashion from a central point for up to several (Genney et al., 2006). metres (Ogawa, 1977). Recent analysis of vegetative mycelia from fruiting bodies indicate that a single shiro of Direct observation of ectomycorrhizal T. matsutake can comprise multiple mycelia (Murata et al., 2005); however, the distribution of these mycelia within the mycelia in the field shiro remains unclear. The indeterminate filamentous nature of fungal hyphae, coupled with the complex nature of soil fungal communities Observations of mycelia in microcosms (Fig. 1) and difficulties in identifying fungal mycelia in the absence of fruiting bodies, makes direct observation and Our understanding of the physiological activities of ECM identification of fungal mycelia in soil virtually impossible, mycelia in soil has been built largely on microcosm studies. and ECM fungi are no exception in this regard (Bridge & The most widely used microcosm system comprises a thin Spooner, 2001; Cairney, 2005). Some higher fungi produce layer of milled peat or soil between two Perspex sheets macroscopic linear mycelial aggregates (= rhizomorphs sensu (Duddridge et al., 1980), or a similar construction (Fig. 1). Cairney et al., 1991) during growth through soil. In the case Such microcosms have been used to elegantly demonstrate of certain saprotrophic fungi, robust rhizomorphs are the roles of ECM mycelia in the absorption and transloca- produced within the soil À litter interface, and this has tion of water, phosphorus and nitrogen from soil to the facilitated excavation, direct measurement and mapping of plant host (Duddridge et al., 1980; Finlay & Read, 1986a, b; more-or-less intact mycelial systems (see Thompson, 1984). Finlay et al., 1988, 1989; Ek et al., 1994; Andersson et al., Although many ECM fungi form rhizomorphs, these are 1996; Timonen et al., 1996; Ek, 1997), along with establish- typically diminutive, and thus direct observations of ECM ing the mycelia as important conduits for carbon movement mycelia in soil have been largely confined to those produced from tree hosts into soil (Finlay & Read, 1986a, b; Ek, 1997; within a few centimetres of ECM root tips or fruiting bodies. Leake et al., 2001; Mahmood et al., 2001; Wu et al., 2002; From investigations of this nature, it is evident that different Heinonsalo et al., 2004; Rosling et al., 2004). The micro- ECM fungi produce different amounts of soil-borne myce- cosms have also been pivotal in identifying potential pat- lium, and that the propensity for mycelia to differentiate to terns and processes involved in the growth, development form rhizomorphs also varies considerably (reviewed by and differentiation of ECM mycelial systems (Read, 1992; Agerer, 2001). Mycelia that remain nonrhizomorphic are Donnelly et al., 2004), along with the likely influence of thought to reflect a limited ability to explore surrounding edaphic changes such as pH (Erland et al., 1990; Ek et al., soil, while mycelia that comprise highly differentiated rhi- 1994; Mahmood et al., 2001). An important observation zomorphs are regarded as more adapted to long-distance here has been the foraging behaviour of ECM mycelia in soil exploration (Agerer, 2001). Between the two extremes are a and, in particular, their abilities to densely colonize discrete range of more-or-less differentiated mycelial types that patches of various organic substrates or necromass from facilitate medium-distance exploration in soil (Agerer, which they can effect nutrient mobilization (Finlay & Read, 2001). According to Agerer (2001), the most highly differ- 1986a, b; Carleton & Read, 1991; Bending & Read, 1995a, b; entiated rhizomorphs can explore ‘up to several decimetres’ Perez-Moreno´ & Read, 2000, 2001a, b; Leake et al., 2001; from the root surface in soil. Difficulties in excavating even Lilleskov & Bruns, 2003; Donnelly et al., 2004; Wallander & the most robust ECM rhizomorph systems in soil make it Pallon, 2005). Microcosms have also been used to investigate difficult to determine the extent to which this underesti- factors such as ECM mycelial longevity and temporal mates the exploration potential of ECM mycelia in soil, but changes in elemental composition (Downes et al., 1992; nonetheless this classification scheme represents a useful Wallander & Pallon, 2005), interactions with soil minerals way to categorize ECM fungi based on the potential (Wallander et al., 2002), responses of mycelia to elevated

ecological relevance of their mycelial systems. atmospheric CO2 concentrations (e.g. Rouhier & Read,

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1998, 1999; Fransson et al., 2005), spatial heterogeneity in strongly affect ECM mycelial growth (Erland et al., 1990; enzyme and gene expression in mycelial systems (Timonen Smith & Read, 1997), such estimation must be viewed with a & Sen, 1998; Wright et al., 2005), and to quantify ECM degree of caution. Attention must also be drawn to the fact myelial area, density, biomass and elemental content in soil that, in most cases, microcosms reflect mycelial growth of a (Ek, 1997; Schubert et al., 2003; Agerer & Raidl, 2004; single ECM fungus in the absence of competing ECM or Donnelly et al., 2004; Hagerberg et al., 2005). other macro fungi. Where competing ECM fungi have been Although microcosm-based studies have facilitated sig- coinoculated in microcosms, it is clear that mycelial devel- nificant advances in our appreciation of the activities of opment can be altered significantly (Wu et al., 1999). While ECM mycelia in soil, there is a danger that they may have the outcomes of interactions appear to vary, largely accord- painted a somewhat unrealistic picture of the extent of ECM ing to relative carbon availability to the individual fungi mycelial system development in the field. Images of ECM (Lindahl, 2000; Lindahl et al., 2001), it is further evident that plants in microcosms generally portray a seedling with a the presence of saprotrophic basidiomycete mycelia can small root system and an ECM mycelial system that radiates influence the growth and development of ECM mycelia in throughout much of the microcosm soil, exploring an area microcosms (Lindahl et al., 1999, 2001; Leake et al., 2001, of soil that is several times that occupied by the root system 2002). Because mycelia of different taxa may preferentially (Fig. 1). The ‘extensive soil-colonizing vegetative mycelium’ grow at different depths in the soil (Lindahl et al., 1999), the produced in microcosms is viewed as representing an ideal largely two-dimensional nature of the microcosms will model of ECM systems in forest soil (Sen, 2000); however, reduce the potential for different mycelia to avoid competi- such extrapolation needs to be undertaken with caution for tion by vertical stratification of growth (Cairney, 2005). Care several reasons. First, the extent of mycelial development in must therefore be taken when attempting to extrapolate more-or-less homogenous milled or sieved microcosm sub- outcomes of competitive interactions between mycelia in strates is unlikely to reflect that through the heterogeneous microcosms to the field. matrix of natural forest soil. Indeed, as highlighted by Smith & Read (1997), the intensive hyphal colonization observed Estimation of mycelial biomass in the field in patches of introduced organic matter (e.g. Finlay & Read, 1986a, b; Bending & Read, 1995b; Perez-Moreno´ & Read, Given the apparent importance of ECM mycelia in forest 2000; Leake et al., 2001) may be more representative of carbon and nutrient cycles, an understanding of their mycelial growth in forest soil than the extensive mycelial belowground biomass is pivotal to fully appreciating their exploration that occurs through the homogeneous micro- contributions to biogeochemical processes. While estimates cosm substrates. The largely two-dimensional nature of the of mycelial biomass in simple nonsoil substrates have been microcosms means that exploration of the substrate is obtained in the laboratory (e.g. Colpaert et al., 1992; effectively on a single plane, and thus the distance that Rousseau et al., 1994), determining ECM mycelial biomass mycelium grows from the root system is likely to be greater in field soil has, until recently, remained a vexed issue. than would be the case for the equivalent mycelial biomass Hogberg¨ & Hogberg¨ (2002) adopted an indirect method in a three-dimensional soil mass. Even where three-dimen- that involved tree girdling, soil fumigation and measure- sional microcosms have been used (e.g. Coutts & Nicholl, ments of dissolved organic carbon to estimate that ECM 1990), however, observations of mycelia rely on growth at mycelium accounts for 4 32% of the total microbial the interface between the soil and the transparent micro- biomass in a boreal forest, while estimates have also been cosm window, and, as such, are unlikely to reflect growth of derived by long-term incubation of soil in the laboratory. the mycelia within the soil column. Add to this the lack of Incubation in the absence of a host plant means that ECM vertical stratification of soil (see below) in the microcosms mycelia should degrade during the incubation period, and (although the rudimentary soil profile reconstruction of thus the difference between fungal biomass, estimated by Heinonsalo et al. (2004) partially addressed this), along with ergosterol or phospholipid fatty acid (PLFA) analysis, before the small size of seedlings, for which the carbon balance and and after incubation, is used as an estimate of ECM fungal patterns of carbon allocation are likely to be dissimilar to biomass (Wallander et al., 2004). Such studies indicate that that of mature trees in the field (Ericsson et al., 1996), and ECM mycelial biomass decreases with soil depth (Ba˚ath˚ the relative growth of mycelia in microcosms may not be et al., 2004; Wallander et al., 2004). much of a guide to mycelia in the field. Hyphal ingrowth bags offer a more direct method for Despite these limitations, rates of growth of ECM mycelia analysis of ECM mycelial growth and biomass in soil. These in microcosms (Read, 1992) have been used to estimate rates are nylon mesh (50 mm) bags containing acid-washed sand of mycelial expansion in the field (e.g. Bonello et al., 1998; that are buried in soil and later retrieved for analysis of total Sawyer et al., 1999). With the above in mind, and the fact fungal content by ergosterol or PLFA analysis (Wallander that the nature of the substrate and edaphic conditions can et al., 2001). The absence of organic matter in the sand

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substrate is thought to select for mycelia of ECM fungi over increase ECM mycelial biomass (Clemmensen et al., 2006). saprotrophs, because ECM fungi have a carbon source from The extent to which these observations reflect altered their tree hosts (Wallander et al., 2001). Comparison of mycelium production per se, or a shift in the ECM fungal mycelial biomass in hyphal ingrowth bags from trenched community towards taxa that produce more/less soil-borne plots (designed to sever ECM roots from their tree hosts, mycelium, however, remains unclear. It is also evident that and so exclude ECM mycelia from the plots) with that from ECM mycelial production can be influenced by temperature untrenched plots suggests that some 85–90% of the myce- (Clemmensen et al., 2006), and that mycelia can grow in lium that colonizes the sand-filled bags in Swedish forest response to certain soil minerals (Hagerberg & Wallander, soils is ECM (Wallander et al., 2001; Hagerberg & Wallander, 2002; Hagerberg et al., 2003) and can contribute to the 2002; Nilsson & Wallander, 2003). This is supported by d13C mobilization of minerals such as apatite (Wallander et al., values derived from the bags (Wallander et al., 2001), along 2002, 2003). with molecular analysis of the fungal taxa present as mycelia in hyphal ingrowth bags from a range of forest habitats Analysis of ECM communities by direct (Wallander et al., 2003; Bastias et al., 2006; Kjller, 2006 and DNA extraction see below). The method relies upon the assumptions that the In recent years methods have been developed to allow use of acid-washed sand provides a reasonable approxima- analysis of microbial communities by direct nucleic acid tion of mycelial growth in native soil, and that there is no extraction from soil. These techniques include cloning, turnover of mycelium during the period in which bags are denaturing gradient gel electrophoresis (DGGE), tempera- buried in soil (Wallander et al., 2001; Hendricks et al., 2006). ture gradient gel electrophoresis (TGGE) and terminal Recent data suggest that ECM mycelial colonization of bags restriction fragment length polymorphism (T-RFLP). The containing acid-washed sand can be considerably reduced technical considerations and limitations associated with compared with that of native soil, indicating that the former these approaches in the context of both general soil (Ander- may underestimate ECM biomass in the surrounding soil, a son & Cairney, 2004; Bidartondo & Gardes, 2005) and ECM fact that may be exacerbated if mycelial turnover occurs (Anderson, 2006) fungal communities have been reviewed during the burial period (Hendricks et al., 2006). The use of recently and thus will not be considered here. soil as a substrate in ingrowth bags, however, requires that there is relatively low fungal biomass in the soil (Wallander, Direct DNA extraction from soil 2006), and further work on the influence of substrate is clearly required. It has also been suggested that ingrowth Recent investigations based on direct DNA extraction from bags may select for certain ECM fungal taxa that are able to soil have yielded information on the diversity and distribu- colonize the substrate rapidly at the expense of slower- tion of ECM mycelia in soil (Table 1). Although they did not growing taxa (Wallander et al., 2003). target ECM fungi specifically, several studies identified Despite the various questions regarding the efficacy of mycelia of ECM fungi as being present in the general fungal ingrowth bags for investigation of ECM mycelia, this assemblages of forest soils (Chen & Cairney, 2002; Anderson method remains the most direct for estimation of ECM et al., 2003; Landeweert et al., 2003a). Given that the mycelial biomass in the field. Work of this nature indicates majority of ECM fungi are basidiomycetes (Smith & Read, that ECM fungal biomass in coniferous forests can be of the 1997), basidiomycete-specific PCR primers have been used order of 100–600 kg haÀ1 and that it comprises a substantial to increase the detection of ECM mycelia in soil DNA part of the soil fungal biomass (Wallander et al., 2001, 2004; extracts. Using this method, Landeweert et al. (2003a) Hagerberg et al., 2003; Hendricks et al., 2006). Growth of reported vertical stratification of certain ECM fungi in the ECM mycelium appears to be seasonal, with greatest activity soil profile, while Smit et al. (2003) demonstrated that in autumn and little or no growth during winter (Wallander removal of litter and humus layers from a pine plantation et al., 2001; Hagerberg & Wallander, 2002). ECM mycelial increased the diversity of ECM mycelia. However, the use biomass has also been shown to vary with soil depth and of basidiomycete-specific primers for the analysis of host tree species, being generally greater in the upper part of ECM mycelial communities has been cautioned (Anderson, the soil profile than in the underlying soil and correlated 2006), because the diversity (e.g. Vralstad˚ et al., 2002; with the distribution of tree roots in the profile (Wallander Tedersoo et al., 2006) and abundance (e.g. Koide et al., et al., 2004; Goransson¨ et al., 2006). It is also evident that 2005a) of ascomycetes in ECM root-tip communities is ECM mycelial biomass in forest soil can be negatively often high. influenced by soil nutrient, particularly nitrogen, status An alternative means of targeting ECM fungi in DNA (Nilsson & Wallander, 2003; Nilsson, 2004; Nilsson et al., extracted directly from soil is T-RFLP analysis and inter- 2005; Hendricks et al., 2006). In contrast, fertilization of rogation of a database containing reference terminal frag- arctic tundra has recently been shown to significantly ments of known ECM fungi from the site. This has

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Table 1. Investigations of soil ECM mycelial communities using direct DNA extraction and molecular analysis methods Forest type DNA extracted from Analysis method Target fungal group Reference Larix kaempferi forest Soil and ECM roots Simple sequence repeat analysis Suillus grevillei Zhou et al. (2001b) Sclerophyll forest Soil Cloning/sequencing Soil fungi Chen & Cairney (2002) Pinus resinosa plantation Soil T-RFLP ECM Dickie et al. (2002) Pinus pinaster forest Soil Competitive PCR Hebeloma cylindrosporum Guidot et al. (2002) Pinus sylvestris forest Soil DGGE/sequencing Soil fungi Anderson et al. (2003) Coniferous forest Soil Cloning/sequencing Basidiomycetes Landeweert et al. (2003a) Pinus sylvestris plantation Soil DGGE and cloning/sequencing Basidiomycetes Smit et al. (2003) Pinus taeda plantation Soil T-RFLP Basidiomycetes Edwards et al. (2004) Pinus pinaster forest Soil Competitive PCR Hebeloma cylindrosporum Guidot et al. (2004) Pinus resinosa plantation Soil and ECM roots T-RFLP ECM Koide et al. (2005a) Pinus resinosa plantation Soil and ECM roots T-RFLP ECM Koide et al. (2005b) Pinus sylvestris forest Soil and ECM roots DGGE/sequencing Basidiomycetes Landeweert et al. (2005) Wet sclerophyll forest Hyphal ingrowth bags DGGE and cloning/sequencing ECM Bastias et al. (2006) Pinus sylvestris plantation Soil and ECM roots T-RFLP ECM Genney et al. (2006) Fagus sylvatica forest Hyphal ingrowth bags Cloning/sequencing ECM Kjller (2006) Picea abies plantation Hyphal ingrowth bags DGGE/sequencing ECM Korkama et al. (2007) Pinus sylvestris forest Soil T-RFLP and cloning/sequencing ECM and saprotrophic fungi Lindahl et al. (2007)

facilitated confirmation of the vertical stratification of ECM Even where individual mycelia of ECM fungal taxa can be mycelial communities in the soil profile (Dickie et al., 2002; identified in soil, observations are restricted to determining Genney et al., 2006). More importantly, Koide et al. (2005b) only the presence or absence of the mycelium in a sampled demonstrated that positive or negative interspecific interac- soil volume, and the methods provide no information on tions can influence the distribution of ECM mycelia in soil mycelial density. Competitive PCR, however, offers a means and that such interactions can vary according to soil of achieving this goal. Indeed, Guidot et al. (2002, 2004) conditions. have used this approach successfully to show that soil-borne T-RFLP analysis of DNA extracted from multiple adjacent mycelia of Hebeloma cylindrosporum are ephemeral, extend soil cores allowed Edwards et al. (2004) to estimate the for o 50 cm, and decrease in density with increasing minimum width of mycelial patches of several ECM taxa in a distance from the base of basidiomes. horizontal plane, the widest patch-size recorded being A fundamental assumption in the interpretation of data 160 cm for Thelephora terrestris. Moreover, Genney et al. generated by direct DNA extraction from soil is that the taxa (2006) coupled T-RFLP analysis with a soil-slicing approach identified are present as mycelia rather than as spores or to investigate the distribution of ECM mycelia in a volume other resting propagules. Although approaches such as the of soil (800 cm3) at a fine scale. Mycelial patches of indivi- collection of soil samples after basidiome production has dual ECM taxa were variously shown to occupy different ceased (Dickie et al., 2002) have been used to minimize the volumes up to a maximum of 312 cm3 for Cadophora likely detection of spore DNA, the fact that DNA from a finlandica. A combination of T-RFLP analysis and cloning small number of H. cylindrosporum spores can be detected has also been used to demonstrate vertical separation of in soil DNA extracts (Guidot et al., 2004) demonstrates ECM and saprotrophic mycelia in a forest-soil profile the potential for spore DNA to be amplified. Given that (Lindahl et al., 2007). While each of these studies demon- dormant propagules are likely to contain little RNA relative strated the potential distribution of mycelia of various ECM to active mycelia, RNA-based approaches (e.g. Anderson & species in forest soil, none considered the distribution Parkin, 2006) may provide a more robust means to detect of individual mycelia ( = genet) of the species. Zhou et al. active ECM mycelia in soil in the future. (2001a, b), however, have demonstrated that simple se- The importance of analysing ECM fungal communities quence repeat (SSR) markers can be applied to investigate by direct DNA extraction from soil is emphasized by the lack the distribution of individual mycelia of Suillus grevillei of congruence between community structures inferred from following direct DNA extraction from soil. With the increas- root-tip and soil-based analyses (Koide et al., 2005a; Land- ing development of SSR markers for ECM fungi (e.g. eweert et al., 2005). Furthermore, it is evident that spatial Bergemann & Miller, 2002; Kretzer et al., 2004; Hitchcock segregation can exist between root tips and mycelia of et al., 2006), information regarding the distribution of individual ECM taxa in the soil profile, with abundant individual mycelia within the soil volume should increase mycelium of some species occurring in the absence of markedly in the near future. infected roots tips (Genney et al., 2006). Indeed, the same

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appears to be true for individual mycelia of S. grevillei (Zhou independent measurements directly. Correlation of both et al., 2001b). relative abundance (community evenness) and species identity (species richness) is important for determining the mycelial biomass of individual ECM species and the re- DNA extraction from hyphal ingrowth bags sponse of individual ECM taxa in manipulative ﬁeld experi- Hyphal ingrowth bags, in conjunction with ergosterol and/ ments. This approach is common in root tip-based or PLFA analysis, has been the main method used to community studies of ECM fungi, although the limitations measure mycelial growth and biomass in forest soil (see of root tip-based measurements of relative abundance have above). More recently, however, the approach has been been previously discussed (Taylor, 2002). While determina- combined with direct DNA extraction from the ingrowth- tion of the relative abundance of mycelia of individual ECM bag substrate and molecular methods to identify the mycelia species is yet to be achieved, this should be possible in the of ECM fungal taxa colonizing the bags. Regardless of the future by combining quantitative molecular methods such molecular method used, these studies indicated that as competitive PCR (e.g. Guidot et al., 2002, 2004) or real- 83–91% of taxa colonizing hyphal ingrowth bags in a wet time quantitative PCR (Landeweert et al., 2003b; Schubert sclerophyll forest (Bastias et al., 2006), a Fagus sylvatica et al., 2003; Raidl et al., 2005) with the hyphal ingrowth-bag forest (Kjller, 2006) or a Picea abies plantation (Korkama approach. Given the known variation in the growth form of et al., 2007) were ECM fungi, which is broadly similar to mycelial systems of different ECM species (Agerer, 2001), hyphal ingrowth bag-based estimates of ECM fungal bio- this will be an important future development in the use of mass in Swedish forest soils using d13C measurements and hyphal ingrowth bags. trenching (Wallander et al., 2001; Hagerberg & Wallander, 2002; Nilsson & Wallander, 2003). ECM mycelia and climate change The hyphal ingrowth-bag approach has been successfully used in conjunction with DGGE to investigate the effects of The importance of understanding the diversity, extent and plant host and forest management practices on ECM dynamics of ECM mycelial systems in forest soils is empha- mycelial communities. Thus, Korkama et al. (2007) demon- sized by current interest in belowground responses to

strated that fast-growing Picea abies clones support different elevated atmospheric CO2 in the context of global climate ECM fungal mycelial communities from slower-growing change (Pendall et al., 2004) and the need to understand clones, with Atheliaceae taxa more abundant in the former. both plant and ECM fungal responses (Alberton et al., Bastias et al. (2006) used this method to establish that long- 2005). ECM fungal mycelia can comprise 80% of the total term repeated prescribed burning of wet sclerophyll forest fungal biomass and 30% of the microbial biomass in some can signiﬁcantly alter the composition of mycelial commu- forest soils (Wallander et al., 2001, 2003; Hogberg¨ & nities of ECM fungi in the upper 10 cm of the soil proﬁle. Hogberg,¨ 2002), with carbon allocation to ECM fungi Cloning and sequencing of DNA extracted from the in- estimated to be as much as 22% of net primary production growth bags further suggested that frequent burning had a (Hobbie, 2006). ECM fungi are thus an important compo- strong negative effect on Cortinariaceae and a positive effect nent of forest carbon cycles, and the effects of elevated

on Thelephoraceae taxa (Bastias et al., 2006). Comparison of atmospheric CO2 on these fungi have deservedly received ECM fungi colonizing hyphal ingrowth bags with those recent attention. Much of this work has focused on interac-

species colonizing root tips adjacent to the ingrowth bags tions between CO2 concentration and root colonization by in a Fagus sylvatica forest showed that Boletoid species ECM fungi. With one exception (Rouhier & Read, 1999), occurred more frequently as mycelia than in root tips, in such investigations have reported increased percentage root

contrast to Russuliod and Cortinarius spp., for which the colonization by ECM fungi under elevated atmospheric CO2 opposite was true (Kjller, 2006). It should be noted that, in conditions (Norby et al., 1987; Ineichen et al., 1995; each of these investigations, only sand was used as substrate Berntson et al., 1997; Godbold et al., 1997; Tingey et al., in the ingrowth bags. Because the nature of the substrate can 1997; Rouhier & Read, 1998; Walker et al., 1998; Kasurinen inﬂuence mycelial colonization of hyphal ingrowth bags et al., 2005).

(Hendricks et al., 2006), it may also bias community Elevated atmospheric CO2 has also been shown to alter composition. To date there is no published comparison ECM fungal root-tip community structure in growth- between mycelia of ECM fungi colonizing sand-filled hyphal chamber experiments (Godbold & Berntson, 1997; Godbold ingrowth bags with mycelia in the surrounding soil. et al., 1997; Rey & Jarvis, 1997; Rygiewicz et al., 2000) and A combination of PLFA analysis and molecular ap- in the field (Fransson et al., 2001; Kasurinen et al., 2005). In proaches allows determination of both mycelial biomass one such experiment, a change in ECM root-tip community and ECM species identity in hyphal ingrowth bags (Korka- composition in favour of morphotypes that appeared to ma et al., 2007), but it is difficult to correlate these two produce emanating hyphae and/or rhizomorphs was noted

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Table 2. Responses of ECM fungal mycelia to elevated atmospheric CO2 concentrations Fungus Host tree Experimental system Mycelial response Reference Hebeloma crustuliniforme Pinus sylvestris Pot experiment Increased mycelial biomass (300%) Fransson et al. (2005) Laccaria bicolor Pinus sylvestris Petri-dish microcosm No effect on hyphal length Gorissen & Kuyper (2000) Paxillus involutus Pinus sylvestris Perspex microcosm Increased mycelial spread (4 400%) Rouhier & Read (1998) Paxillus involutus Betula pendula Perspex microcosm Increased mycelial spread (30%) Rouhier & Read (1999) Pisolithus tinctorius Pinus sylvestris Petri-dish microcosm Increased mycelial biomass (200%) Ineichen et al. (1995) Suillus bovinus Pinus sylvestris Perspex microcosm Increased mycelial spread (200%) Rouhier & Read (1998) Suillus bovinus Pinus sylvestris Petri-dish microcosm No effect on hyphal length Gorissen & Kuyper (2000) ECM mycelial community Betula pendula Field experiment No effect on mycelial biomass Kasurinen et al. (2005) (hyphal ingrowth bags)

under elevated atmospheric CO2 conditions (Godbold & forest ecosystems, where they play crucial roles in nutrient Bernston, 1997; Godbold et al., 1997). Because no attempt and carbon cycling processes. Our appreciation of the roles was made to quantify soil-borne mycelia, however, this of ECM fungi in these processes is hampered by a funda- provides no direct information on the mycelial response of mental lack of understanding of their mycelial systems in ECM fungi under these conditions. Several laboratory-based soil. Much of the currently available information on the microcosm experiments have been undertaken with a view diversity, distribution and dynamics of ECM fungi is derived to determining the responses of ECM mycelia to elevated from investigations of fruiting bodies and ECM root tips,

atmospheric CO2 (Table 2). While Gorissen & Kuyper and provides only limited insights into soil-borne ECM (2000) observed no significant increase in mycelial produc- mycelial communities. Information derived from micro- tion, other studies have reported increases in mycelial cosm studies, while unarguably enhancing our understand- biomass or spread of up to 400% (Ineichen et al., 1995; ing of ECM mycelial functioning, cannot necessarily be Rouhier & Read, 1998, 1999; Fransson et al., 2005). It should extrapolated directly to mycelia in the field, emphasizing be emphasized that these investigations were conducted in the necessity for direct analysis of soil-borne mycelia of simplified microcosm systems (see above) using seedlings ECM fungi in situ. Molecular approaches, combined with inoculated with a single ECM fungus. As such, the observed new experimental approaches, such as hyphal ingrowth responses may not reflect the responses of ECM mycelia to bags, facilitate analysis of ECM fungal communities from

elevated atmospheric CO2 in the ﬁeld, highlighting the need the perspective of mycelia in soil. Furthermore, they have for ﬁeld-based investigations. Although elevated atmo- the potential to increase our understanding of the distribu-

spheric CO2 was found to alter the structure of a basidio- tion of individual mycelia and so enhance our appreciation mycete mycelial community (that included ECM fungi) in a of the physical and physiological continuity of mycelial scrub oak forest (Klamer et al., 2002), to date only one such systems in soil. Such information will give unprecedented study has focused on ECM fungi. By analysing PLFAs from insights into the functioning of ECM mycelia at the ecosys- hyphal ingrowth bags buried under Betula pendula trees in tem level and will assume particular importance when open-top chambers, Kasurinen et al. (2005) established that considering the responses of ECM fungi to future perturba-

elevated atmospheric CO2 had no significant effect on tions, particularly in the context of land-use changes and mycelial production by ECM fungi. global climate change. The inability to generalize based on this single field experiment notwithstanding, these data relate only to the response of the ECM mycelial community and there- Acknowledgements fore provide no information on responses of individual This work was supported by an Australian Research Council ECM species to elevated atmospheric CO2 in the field. Linkage International Awards grant (LX0455012) and a Further studies on how mycelial systems of ECM fungi Macaulay Development Trust collaboration grant. I.C.A. respond to elevated atmospheric CO2 concentrations will receives funding from the Scottish Executive Environment be crucial to modelling and understanding carbon dynamics and Rural Affairs Department. in complex forest ecosystems under future climate-change scenarios. References Conclusions Agerer R (1987–2002) Colour Atlas of Ectomycorrhizae. Einhorn- Verlag, Munich. Soil-borne mycelia of ECM fungi are a dominant and Agerer R (2001) Exploration types of ectomycorrhizae. A important component of soil microbial communities in proposal to classify ectomycorrhizal mycelial systems

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Home » Nature What Are Mycelia in Microbiology? By Andrea Becker; Updated April 25, 2017

The fungi kingdom sits on the border between plants and animals and between micro- and macro-biology. The mycelium, plural mycelia, exemplifies how the microscopic elements of fungi can combine to form a larger whole. Mycelia are the diffuse vegetative parts of multicellular filamentous fungi. Filamentous fungi can be divided into microfungi and macrofungi, but the mycelia of both groups have similar form and function. They are made up of a network of threads that are often too fine to be seen by the naked eye, known as hyphae. Networks of Hyphae Hyphae are compartmentalized tubes that grow out into food sources in order to digest and absorb nutrients. Fungi are heterotrophs, they need to digest other organisms to get their energy, and they can digest tough foods, such as dead trees and insect carapaces. Hyphae grow out from the end of the tube and can branch, forming networks of threads, each not more than one hundredth of a millimeter (0.0004 inches) in diameter. In total, this network is known as the mycelium. Hyphae are why the mold on your bread looks fuzzy.

Mycelia Growth As a mycelium grows out into a substrate it excretes enzymes at the tips of its hyphae that digest the substrate into a form that can be absorbed by the fungus. The more nutrients are in the substrate, the more branches the mycelia forms to take advantage of the food source. Mycellia move out from the location of the original fungal spore, but since it uses up all of the nutrients in the center, the middle of the circle gets cannibalized, causing a ring-like pattern recognizable in fairy rings and ringworm infections.

Examples of Microfungi Mycellia The ability of the mycelium to spread through a substrate as they digest it makes ﬁlamentous microfungi both important decomposers and parasites. There are over 13,000 species that have been identiﬁed in the United States, but that likely represents only a tiny fraction of the species out there. The mycelia of Phytophthora infestans spreads through potato tubers, rotting them, the cause of the Irish potato famine. The mycelia of Trichoderma reesei, a fungus that breaks down dead plant matter, excretes three different types of cellulase to fully digest the cellulose in its food supply.

When Mycelia Become Macroscopic The mycelia of most fungi are microscopic, but there are times when mycelia form larger conglomerate structures. The structure that is most familiar is the fruiting body, or mushroom, a reproductive structure used to disseminate spores to new environments. Fungal mycelia can also form rhizomorphs, or cords of bundled hyphae, and sclerotia, or structures that anchor the fungus and store nutrients to use during adverse conditions. While the individual hyphae are microscopic, a single honey mushroom is actually the largest and oldest known living organism, spread over 890 hectares (2,200 acres) of ground and dubbed the humongous fungus.

References Biology Online: Mycelium Australian National Herbarium: The Mycelium Microbiology Online: Fungi US Geological Survey: Microfungi: Molds, Mildews, Rusts, and Smuts Fungal Biology: Process of Extracellular Digestion Microbe World: Fungi

About the Author Based in Wenatchee, Wash., Andrea Becker specializes in biology, ecology and environmental sciences. She has written peer-reviewed articles in the "Journal of Wildlife Management," policy documents,and educational materials. She holds a Master of Science in wildlife management from Iowa State University. She was once charged by a grizzly bear while on the job.