Current Investigations of Fungal 

Ectomycorrhizal Communities in the 

Sierra National Forest1 

Thomas D. Bruns,2 Annette M. Kretzer,

3 Thomas R. 

Horton,4 Eric A-D. Stendell,5 Martin I. Bidartondo,

6 

and Timothy M. Szaro7 

Abstract 

Progress on two main studies on fungal ectomycorrhizal communities in the Sierra 

National Forest is discussed. One study examined the short-term effects of ground fire 

on the ectomycorrhizal community and the other examined the ectomycorrhizal 

associates of snow plant (Sarcodes sanguinea). In the ground-fire study we found that a 

large initial reduction in ectomycorrhizal biomass is caused primarily by combustion 

of the upper organic layers; prefire dominants in the Russulaceae and Thelephoraceae 

are dramatically reduced; and species at greater depths appear to survive the fire. We 

speculate that fire causes a short-term increase in species evenness. In the field 

portion of the Sarcodes study, we found that Sarcodes is specialized on a single 

mycorrhizal associate, Rhizopogon ellenae; the presence of Sarcodes is associated with 

dense islands of R. ellenae; R. ellenae appears to be a minor below-ground component 

of the red fir (Abies magnifica) community in locations near but not immediately 

adjacent to Sarcodes plants; and the Abies magnifica ectomycorrhizal community is 

dominated by members of the Russulaceae and Thelephoraceae. From studies of 

Sarcodes-R. ellenae interactions under laboratory conditions, we report that seed 

germination is stimulated by isolates of R. ellenae, isolates of R. ellenae derived from 

Sarcodes and Abies roots are capable of forming mycorrhizae on pine (Pinus) roots, and 

we have achieved initial success in establishing a tripartite Sarcodes-Rhizopogon-Pinus 

association under laboratory conditions. 

Ectomycorrhizae (EM) are dual organs composed of the fine roots of plants 

and fungal mycelia. They are the primary interface through which most temperate 

forest trees receive their mineral nutrients. All members of the Pinaceae require 

these mutualistic fungi for normal growth and survival (Smith and Read 1997). 

The diversity of EM fungi is extremely high. Over 6,000 species have been 

described (Molina and others 1992) and, because of the current state of our 

taxonomic knowledge, this probably represents a gross underestimation of the 

total number. Even at a local scale, diversity is very high. In single-species pine 

stands of approximately 0.1 ha, 15 to 35 species of EM fungi are typically reported, 

single soil cores often contain several species (Eberhart and others 1996), and 

even adjacent root tips are frequently colonized by different fungi (Bruns 1995). 

In spite of the importance of EM fungi, little is known about the structure of 

the complex communities that they form or the functional differences among the 

component species. Until recently, almost no quantitative descriptions of the 

abundance of EM species on roots were reported. This lack of information was 

largely caused by the difficulty in identifying fungal species in their vegetative 

states (that is, as mycorrhizae or mycelia). With the advent of the polymerase 

chain reaction (PCR), molecular-based methods now make identification much 

1 	An abbreviated version of this 

paper was presented at the 

Symposium on the Kings River 

Sustainable Forest Ecosystems 

Project: Progress and Current 

Status, January 26, 1998, Clovis, 

California. 

2 Professor, Department of Plant and 

Microbial Biology, University of 

California, Berkeley, CA 94720. 

3 Adjunct Professor, Department of 

Environmental and Forest 

Biology, SUNY-ESF, Syracuse, NY 

13210. 

4 Assistant Professor, Department 

of Environmental and Forest 

Biology, SUNY-ESF, Syracuse, NY 

13210. 

5 Student, Department of Plant and 

Microbial Biology, University of 

California, Berkeley, CA 94720. 

6 Graduate Student, Department of 

Plant and Microbial Biology, 

University of California, Berkeley, 

CA 94720. 

7 Technician, Department of Plant 

and Microbial Biology, University 

of California, Berkeley, CA 94720. 

USDA Forest Service Gen. Tech. Rep. PSW-GTR-183. 2002. 83 
Bruns, Kretzer, Horton, Stendell, Bidartondo, Szaro Current Investigations of Fungal Ectomycorrhizal Communities in the Sierra National Forests 

more feasible. As a result, many EM community studies are beginning to be 

reported (Erland 1995, Gardes and Bruns 1996a, Horton and Bruns 1998, Horton 

and others 1998, Horton and others 1999, Kårén and Nylund 1996). 

The long-term goals of all our studies are to examine the structure of EM 

communities in diverse settings, to look for common themes in these structures, 

and to elucidate the functional differences among EM fungi. Our strategy has 

been to focus on a series of much smaller but more researchable questions and to 

assemble results of these studies into a general picture of fungal EM communities. 

Our prior work in coastal pine and Douglas-fir (Pseudotsuga menziesii) forests has 

shown that the species that dominate fruiting can be minor components of the 

below-ground community; mature communities are typically dominated by 

members of the Thelephoraceae and Russulaceae; where roots of Douglas-fir and 

pine or Douglas-fir and manzanita (Arctostaphylos spp.) intermingle, they usually 

are associated with the same fungal species; and a spore-bank guild, dominated 

by members of the Suillineae (primarily Rhizopogon) and members of the 

Ascomycota, is present in forest soils, and these species dominate pine seedlings 

in the first few years after stand-replacing fires (Baar and others 1999, Gardes 

and Bruns 1996a, Horton and Bruns 1998, Horton and others 1998, Horton and 

others in press, Taylor and Bruns 1999). 

In the Sierra National Forest (SNF) we focused on two studies that 

complemented our earlier or ongoing studies elsewhere. In the first, we examined 

the short-term effects of ground fire on the EM community associated with 

ponderosa pine (Stendell and others 1999). The second study focused on EM 

associates of snow plant and on the EM community on red fir (Abies magnifica) 

surrounding snow plants. Sarcodes (fig. 1) is a nonphotosynthetic member of the 

Ericaceae, subfamily Monotropoideae. It is “epiparasitic” in that it obtains all of 

its carbon from surrounding trees via an EM fungus. Early studies in our lab on 

other nonphotosynthetic epiparasitic plants showed that, in contrast to 

photosynthetic plants, virtually all of the nonphotosynthetic plants that we 

studied had highly specific fungal associations. The only apparent exception was 

Figure 1—Sarcodes sanguinea. 

84 USDA Forest Service Gen. Tech. Rep. PSW-GTR-183. 2002. 
Current Investigations of Fungal Ectomycorrhizal Communities in the Sierra National Forests Bruns, Kretzer, Horton, Stendell, Bidartondo, Szaro 

Sarcodes, and because it is a common plant in SNF, we decided to sample it more 

intensively and more locally than in our previous study, which included only 12 

Sarcodes plants (Cullings and others 1996). In the course of sampling we found a 

site where Sarcodes was unusually abundant. This “high-density” Sarcodes site, 

situated in a red fir monoculture, enabled us to examine the EM community 

adjacent to Sarcodes and compare it with that in the surrounding red fir forest. It 

also provided an ideal setting for studying the sizes of fungal clones associated 

with Sarcodes. 

Methods 

Collection locations for fungi and Sarcodes were dispersed throughout the mixedconifer and red fir zones of SNF. Global positioning system (GPS) coordinates 

were recorded for collection sites and plots. Collections of fungal fruitbodies 

were dried and retained in our herbarium, later to be deposited in the University 

of California Herbarium. Larger collections were split, with half of each deposited 

at Oregon State University. Most hypogeous collections were examined and 

identified by Dr. Jim Trappe of Oregon State University. In addition, Rhizopogon 

species in section Amylopogon were identified by either restriction fragment 

length polymorphism (RFLP) or sequence analysis of the internal transcribed 

spacer region (ITS). 

Pine and fir roots were washed from soil cores of 4 X 40 cm. EM were sorted 

by shape and color, freeze-dried, weighed, and saved for molecular analysis. 

Sarcodes roots were collected by digging alongside flowering spikes until the 

rootball was encountered. Portions of the rootball were then removed, washed, 

and freeze-dried or used for isolation of the fungal symbiont into axenic culture. 

EM fungi were identified by molecular analysis as described previously (Bruns 

and others 1998, Gardes and Bruns 1993, Gardes and Bruns1996b). 

Plots for the fire study were located at 36o

58’48"N latitude and 119o8’13"W 

longitude. Six sampling plots were located in small, mature stands of ponderosa 

pine on a south-facing slope of approximately 11o on either side of the SNF 

service road 10S67. North of the road, in an area intended for a prescribed burn, 

three plots were established prior to the burn, and three control plots were 

established south of the road outside the designated burn area. 

Studies of the EM community surrounding individual Sarcodes plants were 

done in a pure red fir stand near the junction of the SNF service roads 9S62 and 

9S10 at 37o

09'06" N latitude and 119o07’50" W longitude. Plants in this plot were 

counted along a 100-m transect, mapped within a 750-m2 grid, and soil cores 

were taken along four 10-m transects centered on individual Sarcodes plants. 

Results and Discussion 

Results from the fire study (Stendell and others 1999) showed that the presence 

of all species was highly patchy in both the prefire samples and postfire control 

plots. Even two cores taken from a single m2 plot often contained entirely 

different species. In spite of this, we observed that members of the Russulaceae 

and Thelephoraceae dominated the community—a pattern shared by all other 

undisturbed California pinaceous communities that we have studied (Gardes 

and Bruns 1996a, Horton and Bruns 1998, Horton and others 1999, Taylor and 

Bruns 1999). The short-term effect of this ground fire on the mycorrhizal 

community was an eightfold reduction in ectomycorrhizal biomass. The 

reduction was directly correlated with incineration of the litter layer and heating 

of the top few cm directly below it. These two regions accounted for the 

overwhelming majority of prefire mycorrhizae. There appeared to be a trend 

toward disproportionate reduction of the most abundant ectomycorrhizal fungi— 

members of the Russulaceae and Thelephoraceae. We speculate that this resulted 

USDA Forest Service Gen. Tech. Rep. PSW-GTR-183. 2002. 85 
Bruns, Kretzer, Horton, Stendell, Bidartondo, Szaro Current Investigations of Fungal Ectomycorrhizal Communities in the Sierra National Forests 

because these fungi are primarily dominant in the organic upper soil layers that 

were destroyed. There appears to be a trend for rare types to be more common at 

greater depths in all communities we have studied. Thus, one of the immediate 

effects of fire may be to increase species evenness in the ectomycorrhizal communities 

by destroying the organic layers where a few species dominate, while preserving the 

deeper layers where species richness is greatest. 

During the summer of 1998, we returned to the fire site and resampled the 

ectomycorrhizal community to see how it had recovered in the third year after 

the fire. Although we will not have the answer to this question for several 

months, we did observe abundant fruiting of Rhizopogon subcaerulescens at the 

site that summer. This is interesting because we knew from our 1-year sample 

that it is one of the species that survived the fire. We also know from our studies 

of the Pt. Reyes wildfire that R. subcaerulescens is one of the most dominant and 

responsive members of the sporebank in fire-disturbed settings (Baar and others 

1999, Horton and others 1998, Taylor and Bruns 1999). It would not be surprising, 

therefore, if its abundance increased as a result of the burn. 

From our sample of 64 Sarcodes, we determined that the only mycorrhizal 

associate for Sarcodes within the SNF and the Tahoe basin is in the Rhizopogon 

ellenae species complex. Previously, we knew that the associate was close to R. 

subcaerulescens, but with a refined ITS sequence database, we discovered that the 

associate is distinct from R. subcaerulescens and closer to R. ellenae. This is 

interesting because it means that snow plant targets a different fungus from its 

close relative, pine drops (Pterospora andromedea—fig. 2); the latter is associated 

with the R. subcaerulescens complex. The fact that these two plants often grow 

within a few meters of each other demonstrates that the plant-fungal specificity 

is based on intrinsic factors of the interaction rather than habitat. It also shows 

that these two fungi, which are difficult to discriminate taxonomically, are not 

redundant; the absence of either would translate into the absence of either 

Sarcodes or Pterospora. 

The identification of R. ellenae as the associate of Sarcodes also revealed the 

confusion within identified Rhizopogon collections. Initially, we thought that R. 

ellenae was rare in the SNF because it did not appear to be included in any of our 

collections from the area. After we applied ITS-RFLP analysis to our collections, 

however, and to many of those housed in the Thiers Herbarium at San Francisco 

State University, we discovered that the names R. subcaerulescens and R. ellenae 

have essentially been applied interchangeably, even though two distinct lineages 

can be identified easily by ITS sequences. 

The soil cores taken along four transects and centered on individual snow 

plants have revealed two interesting results. First, we found that both the 

presence of R. ellenae on fir roots and the number of fir roots are orders of 

magnitude greater near the rootball of Sarcodes than in the surrounding forest. 

This suggests that Sarcodes is either finding dense islands of R. ellenae-Abies 

mycorrhizae, or it is creating them. If the latter is the case, then the relationship 

between snow plant and Rhizopogon does not look strictly parasitic, as we had 

thought, and it opens the question of how this nonphotosynthetic plant is capable 

of stimulating both its fungal associate and the roots of a photosynthetic host. 

Second, we found that R. ellenae is so rare on the roots of Abies 500 cm from 

Sarcodes plants that it is undetected in our sample, and the dominant fungi are 

members of the Russulaceae and Thelephoraceae. This result provides further 

evidence that their dominance is a general pattern in California pinaceous 

ecosystems. 

With the knowledge that all Sarcodes plants were associated with R. ellenae, 

the flower spikes were used as a convenient way to locate sites where the fungus 

was present. We selected plants at the high-density site, mapped them, and 

cultured R. ellenae from their roots. We are now analyzing these isolates to 

determine which, if any, are genetically identical. Because Rhizopogon has no 

86 USDA Forest Service Gen. Tech. Rep. PSW-GTR-183. 2002. 
Current Investigations of Fungal Ectomycorrhizal Communities in the Sierra National Forests Bruns, Kretzer, Horton, Stendell, Bidartondo, Szaro 

Figure 2—Pterospora andromedea. 

known asexual spore state, genetic identity can be achieved only by vegetative 

growth. Therefore, by relating identical genotypes back to their mapped locations 

in the forest, we will be able to tell the size of a given fungal individual. We did 

not know what to expect for a Rhizopogon species, but we did know that Suillus 

species, which are closely related to Rhizopogon, often have individuals that span 

100 m2 or more (Bonello and others 1998, Dahlberg 1997, Dahlberg and Stenlid 

1994, Jacobson and others 1993). Our preliminary results from R. ellenae at the 

dense site suggest that the average individual is small and that more than one 

individual may colonize a single Sarcodes plant. If confirmed, this would suggest 

that the abundance of R. ellenae at this site is the result of recent establishment by 

spore. This may be related to the past history of this site, which was burned after 

thinning about 6 years prior to this study. We know from our other studies that 

spores of several Rhizopogon species respond positively to fire or soil disturbance 

(Baar and others 1999, Horton and others 1998, Taylor and Bruns 1999). 

Recently we have been using cultures of R. ellenae and R. subcaerulescens that 

we isolated from snow plant and pine drops, respectively, to study the 

physiological ecology of the ectomycorrhizal interaction. Our preliminary results 

show that both species are capable of forming mycorrhizae with lodgepole pine 

(Pinus contorta) and bishop pine (Pinus muricata), which agrees with previous 

findings by Molina and Trappe (1994). We also have preliminary evidence that R. 

ellenae stimulates the germination of snow plant seeds. This is the first record of 

in vitro germination for this species; ongoing tests will determine whether 

germination is specific to R. ellenae. Very recently we have succeeded in growing 

USDA Forest Service Gen. Tech. Rep. PSW-GTR-183. 2002. 87 
Bruns, Kretzer, Horton, Stendell, Bidartondo, Szaro Current Investigations of Fungal Ectomycorrhizal Communities in the Sierra National Forests 

Sarcodes in the laboratory in a tripartite system with lodgepole pine and R. 

ellenae. This is the first time a member of the Monotropoideae has successfully 

been grown in the laboratory, and it offers us an opportunity to use this system 

to further study the physiology of these plants. 

A general picture of EM communities is starting to come into focus. They are 

clearly diverse and highly patchy communities, but their structure, at least at the 

family level, appears to be similar across different types of California pinaceous 

forests. Understanding the autecology of individual species is a daunting task, 

but one that is necessary if we wish to understand how these organisms interact 

with plant and animal communities. 

Acknowledgments 

We are grateful to Dan Luomo and Randy Molina for critically reviewing a draft 

manuscript of this paper and to Jim Trappe, Francisco Comacho, Ari Jumpponen, 

Teresa Lebel, Liz Pine, and Eric Peterson for their help with collection and 

identification of hypogeous fungi. Lisa Grubisha of Oregon State University 

assisted with a refined ITS sequencing process. We are also indebted to Dave 

McCandliss for helping us select the site for our fire study and for a 

beautifully executed prescribed burn. Finally, we thank Jared Verner and 

George Steger for arranging a trailer for our use at the Dinkey Creek Work 

station for the past 4 years; none of this work would have been possible 

without it. Financial support was provided in part by Cooperative Agreement 

PSW-94-0023CA with the Pacific Southwest Research Station, USDA Forest 

Service; a USDA forest biology competitive grant; and a grant from the 

ecology program of the National Science Foundation. 

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