Microfungi of Forest Litter From Healthy American
Beech, Fraser Fir, and Eastern Hemlock Stands in Great
Smoky Mountains National Park
Richard E. Baird, Sandra Woolfolk, and Clarence E. Watson
Southeastern Naturalist, Volume 8, Number 4 (2009): 609–630
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2009 SOUTHEASTERN NATURALIST 8(4):609–630
Microfungi of Forest Litter From Healthy American
Beech, Fraser Fir, and Eastern Hemlock Stands in Great
Smoky Mountains National Park
Richard E. Baird1, Sandra Woolfolk1, and Clarence E. Watson2
Abstract - As part of an All Taxa Biodiversity Inventory of the Great Smoky Mountains
National Park, an assemblage of microfungi associated in litter samples from
healthy Fagus grandifolia (American Beech), Abies fraseri (Fraser Fir), and Tsuga
canadensis (Eastern Hemlock) trees was determined in 2005 and 2006. Additionally,
litter samples from the collection sites were assayed for pH, nutrient content,
ash, crude proteins, and levels of organic matter to determine their impact on the
mycobiota. Species richness, diversity, and evenness patterns were evaluated from
the litter samples collected in May, July, and September of each year. A total of 6249
isolates of fungi were obtained, with greater than 90% belonging to the Deuteromycota.
Over 100 species of fungi were identified from litter of the three tree species,
with 55 being new records from the Park. As in previous studies, the most common
fungi isolated from the three tree species were 13 species of Trichoderma during
the two-year study. Other common fungi included Virgaria nigra and Penicillium
spp. Species richness and diversity values pooled across sampling dates and years
were significantly greater from American Beech litter, followed by Eastern Hemlock
and lowest for Fraser Fir. Species richness and diversity values compared by sampling
dates for each year were generally greater in May than July or September, but
evenness values showed a reverse trend for each year. When species richness and
diversity were compared between sampling dates per year and among or by tree species,
significant differences often occurred, but no trends were determined. Data from
the litter tissue assay showed that Fraser Fir, which had the lowest species richness
and diversity, may have been impacted by having significantly lower pH and percent
litter chemical compositions of ash, crude protein, and N than the other tree species.
All other comparisons of species richness were similar.
An All Taxa Biodiversity Inventory (ATBI) in the Great Smoky Mountains
National Park (GSMNP) has been ongoing for all categories of organisms
including macro- and microscopic fungi over the past decade (Baird et al.
2007, Sharkey 2001). In the latter study, the bark of Fagus grandifolia Ehrh.
(American Beech), Tsuga canadensis (L.) Carr. (Eastern Hemlock), and
Abies fraseri (Pursh) Poir. (Fraser Fir) in GSMNP were sampled for the presence
of microfungi. These three tree species were selected for study since
baseline data from healthy stands will soon be lost throughout the Park and
region from introduced exotic pests (Baird et al. 2007). In this study, a total
1Entomology and Plant Pathology Department, Box 9655, Mississippi State University,
Mississippi State, MS 39762. 2Division of Agricultural Sciences and Natural
Resources, Oklahoma State University, 139 Ag Hall, Stillwater, OK 74078. *Corresponding
author - email@example.com.
610 Southeastern Naturalist Vol. 8, No. 4
of 93 different species of fungi were identified from bark of the three tree
species, using selected isolation media.
Species assemblages have been shown to be highly variable within different
climatic regions or zones (e.g., tropical versus temperate) (Cannon and
Sutton 2004). These differences in fungal species not only occur across large
geographical regions, but local differences related to niche can have a dramatic
impact on their diversity. For example, taxa from low elevations can
differ significantly from those collected at higher elevations, but both sites
can occur within the same geographical area (Baird 1986). The differences
in fungal diversity are due to a combination of factors such as variations in
temperature and fl ora between the sites. Factors that infl uence site species
richness include temperature, water relations, available nutrition, pH, plant
host diversity, seasonality, and physical factors (Barron 2003, Cannon and
Sutton 2004). It has also been stated that specific slopes (aspects) will affect
the amounts of local rainfall, temperature, and plant species present. Because
forest ecosystems within the GSMNP vary due to elevation differences
within a small geographical area, many of the factors listed above would be
expected to have an impact on microfungal assemblages.
Forest soils are continually enriched by plant debris derived from aboveground
biomass or forest litter (Fogel 1980). Litter has often been defined
as containing dead leaves, fruits, seeds, and wood ≤5 cm in diameter (Rossman
et al. 1998), but litter can also include unrecognizable plant tissues that
have been degraded (Bills and Polishook 1994). Organisms characteristically
found within the complex litter microhabitat include bacteria, fungi,
bryophytes, yeasts, lichens, and mesofauna (Bier 1963a,b; Bills et al. 2004).
However, fungi were reported to comprise nearly 89% of the living microbial
biomass in deciduous forests of Britain (Frankland 1981). Burges (1965)
stated that over 600 species of anamorphic forming fungi were identified
from forest litter, whereas Watanabe (1994) suggested that over 1200 or
more fungal species colonized dead tissue. Most recently, Cannon and Sutton
(2004) estimated that approximately 23,500 species of fungi occur on dead
plant tissue. These include members of the Ascomycota, Basidiomycota, and
Zygomycota along with their anamorphic taxa contained within the artificial
assemblage Fungi Imperfecti (mitosporic). The majority of mitosporic fungi
identified belonged to the Ascomycota and a smaller percentage to the Basidiomycota.
In a previous study conducted in a Costa Rican rain forest, four
samples of 1-ml sterile, distilled-water suspensions from leaf litter yielded
a total of 1709 isolates (Bills and Polishook 1994). Within these samples,
the most abundant species identified occur in no more than 23% of the total
samples. In another study, repeated over three consecutive years, senescent
and decayed Sugar Maple leaves contained a succession of microfungi that
were consistently identified each year (Kuter 1986). The succession was
partially based on changes due to nutritional succession and weather-pattern
variations throughout each year. Hogg and Hudson (1966) followed the progression
of decay of beech leaf litter over a two-year period and determined
that fungal species increased over time when identified directly from the
2009 R.E. Baird, S. Woolfolk, and C.E. Watson 611
tissues, but densities decreased over the same period using cultural studies
with leaf fragments. Additional research involving surveys of microfungi
from litter of different habitats have been conducted during the last 50 years
and have utilized different sampling and laboratory methods for identifications
(Bandoni 1981; Gremmen 1957; Hayes 1965; Heredia 1993; Hering
1965; Kendrick and Burgess 1962; Polishook et al. 1996; Remacle 1971;
Subramanian and Vittal 1979; Tubaki and Yokoyama 1971, 1973; Visser and
Parkinson 1975; Wildman and Parkinson 1979). The numbers and diversity
of microfungi identified in those studies may have been affected in part by
experimental factors such as temperature for incubation, choice of growth
media (high-nutrition), and the presence of fast-growing contaminates or superficially occurring mycofl ora. When conducting “total inventory” studies
of microfungi, the techniques employed by Bills and Polishook (1994) are
presently accepted as the best procedures for microfungal surveys of litter
(Cannon and Sutton 2004).
Various soil factors infl uence the accuracy of cataloging the diversities
and densities of microfungi during a “total inventory.” These factors include
litter particle size used for processing samples, growth-medium composition,
and growth inhibitors added to media (Cannon and Sutton 2004).
Particle size of litter fragments should be small enough to avoid multiple
colonies arising from the same fragments. For heavily degraded tissues,
specific tissue sizes cannot be readily obtained, and litter weight must then
be used for consistency. Growth media have been shown in many ecological
studies of fungi to affect the isolation frequencies of the microfungi. The
most effective types of media are ones low in nutrients, since hyphal growth
will then be restricted for fast growing fungi (e.g., Rhizopus) and allow the
slow-growing species to be observed (Bills and Polishook 1994, Bills et al.
2004). Lastly, strategies which slow growth of fast-growing fungi, including
incubating colonies at low temperatures and/or the addition of chemicals in
the growth media such as cyclosporin A, selective fungicides (e.g., Botran
for Rhizopus spp.), and antibiotics for bacteria, have proven to allow slowergrowing
fungi to be identified (Baird et al. 1991). The objectives of the study
reported herein were to develop baseline data by cataloging the fungal microfl
ora present in litter samples collected under stands of American Beech,
Eastern Hemlock, and Fraser Fir for the ATBI in GSMNP and to compare select
parameters including date of sampling litter, plant-tissue nutrient levels,
and other chemical component levels that can infl uence species richness.
Materials and Methods
Field collections of litter samples were obtained from American Beech,
Eastern Hemlock, and Fraser Fir stands at most of the locations used previously
for bark samples (Baird et al. 2007). Samples were collected in May,
July, and September of 2005 and 2006 (Table 1). Litter samples were always
taken from beneath trees ≥20 cm in diameter at breast-height 1.3 m above
the ground. Additional criteria for sampling are discussed below.
612 Southeastern Naturalist Vol. 8, No. 4
Table 1. Sampling dates and locations within GSMNP of litter samples collected from three tree species over a two-year period.
Sampling date American Beech Fraser Fir Eastern Hemlock American Beech Fraser Fir Eastern Hemlock
May 20–25 Cataloochee, GSMNP- Clingman’s Dome A Cataloochee, GSMNP- Behind Ball House Mt. Buckley Gabes Mt. Trail
Horse Camp before Big Fork Ridge Trail near SR 441
Little Cataloochee Trail
17S9443951NA 17S3938104N 17S3939633N 17S3959984N 17S3938089N 17S3959133N
17S0273087E 17S0272998E 17S0307911E 17S0294193E 17S0272790E 17S297330E
July 1–3 Beech Gap A Appalachian Trail Beech Gap B Fork Ridge Trail Mt. Sterling A Laurel Falls
17S3946910N 17S3938128N 17S3946822N 17S3939907N 17S3952586N 17S3950395N
17S0300490E 17S0271952E 17S0300636E 17S0278128E 17S0307796E 17S0266283E
Sept 9–10 Beech Forest on New Mt. Buckley Thomas Divide Trail Sugarlands Center Clingman’s Dome B Copeland Ck Area
Found Gap Trail
17S3943462N 17S3938089N 17S3939844N 17S3951831N 17S3938080N 17S3957991N
17S0278482E 17S272790E 17S284566E 17S0270532E 17S0273302E 17S283518E
AUTM NAD27 CONUS
2009 R.E. Baird, S. Woolfolk, and C.E. Watson 613
To determine the diversity and density of the microfungi present in the
litter samples, specific sampling strategies were used for “total inventory”
determination. All samples were collected under closed canopies and consisted
of all forms of tissues including fallen leaves, twigs, and seeds in
different stages of decomposition. On each sampling date, four replicate
trees (healthy) per species were sampled at each location. Healthy trees exhibited
no symptoms of damage or presence of exotic pest and had a canopy
that was considered 90–100% intact. All litter samples were collected from
the north side of each of the trees within 1 m of the boles for uniformity and
was raked unsorted by hand from a 10 cm2 area into large office envelopes.
The litter samples, which included recognizable and unrecognizable tissues
stored in the envelope (500 ml of material), were placed into cold storage
(10 ºC) and returned to the laboratory for processing.
Techniques previously developed by Bills and Polishook (1994) and
Cannon and Sutton (2003) and refined by Bills et al. (2004) were used for
isolation and identification of the microfungi from litter samples from each
location. Using the methods developed by Bills et al. (2004), the plant tissues
were returned to the laboratory and air dried for 3–4 hr. Following drying,
5 g of litter tissue, consisting of all stages of decay, were placed into a sterile
Black & Decker Mixer (Handy Chopper Plus™) and pulverized for 1 min.
The tissue mixture was then washed in a steady stream of sterile distilled
water for 10 min through 2-mm brass prescreens and then through two sterilized
polypropylene mesh filters (Spectra/Mesh 210-μm and 105-μm). After
washing, the 105-μm filter was placed into a 50-ml polystyrene centrifuge
tube, and sterile distilled water was added. The tube was agitated vigorously
for up to 1 min, and the filter was then removed, allowing the particles to
settle. The supernatant was removed, and the particles were rewashed in 50
ml of sterile distilled water and allowed to settle. To ensure a uniform density
of particles for each sample, sterile distilled water was added to all for a
20:1 (v/v) ratio of water/particles. For each tube, the particles were agitated
and resuspended. At this time, 0.1 ml of the suspension was pippetted and
added to each plate containing either CYCL or DRBC media (Bills et al.
2004). From each sample suspension, ten replicate plates per medium were
used. All ingredients and antibiotics for the two media were those previously
reported (Bills and Poolishook 1994). The 0.1-ml particle suspension
was spread over the surface of the agar for each plate (10 x 100 mm) with a
fl amed bent-glass rod. The plates were incubated at room temperature and
12-h photoperiod under artificial lighting. Four days after the suspension
was added to the plates, five colonies per plate were randomly selected and
placed onto potato-dextrose agar (PDA; Difco®, Detroit, MI) and corn-meal
agar media (CMA; Difco®) and stored for later identification using standard
mycological methods (Baird et al. 2004, 2007). Due to the large number
of colonies per sampling period, only a limited number were subcultured,
as suggested previously (Baird et al. 2007, Woolfolk and Inglis 2004).
614 Southeastern Naturalist Vol. 8, No. 4
Identifications were determined for the genera and species isolated. Single
spores of cultures initially identified as Fusarium spp. were transferred to
carnation leaf agar and identified using the classification system of Nelson
et al. (1983). Keys for general identification of fungi were those developed
by Ellis (1971), Sutton (1980), and Barnett and Hunter (1998). In addition,
an unpublished guide and keys to Trichoderma spp. by G. Samuels, USDA/
ARS-Beltsville, MD were used.
Nutrient analyses of litter samples
For all sampling dates, litter samples were evaluated to determine
percent ash content, crude protein, pH, and organic matter as analyzed by
Mississippi State Chemical Laboratory, Mississippi State University. Procedures
were followed using standard methods for nutrient analyses (Horwitz
2000). Total carbon (C) and nitrogen (N) levels and C:N ratios were obtained
using a Fisons NA 1500 NCS analyzer (ThermoQuest Italia, Milan, Italy)
and following Dumas combustion techniques (Baccanti et al. 1993, Bellomonte
et al. 1987, Jones and Case 1990). All procedures for C and N were
conducted at Forest Hydrology Laboratory, Mississippi State University.
Statistical analysis of data
The experimental design was a completely randomized design (CRD)
within each tree species and sampling date. Species richness values (SR)
and species diversity indices (H') were calculated using Shannon-Weaver
index, coefficient of community (CC), and evenness (E) (Stephenson 1989,
Stephenson et al. 2004). Stephenson (1989) provides a thorough description
of all formulas for these indices. Data were further analyzed as series of
combined CRD’s using the GLM procedure of SAS (SAS Institute, Cary,
NC), and means were separated using Fisher’s protected least significant
Over 100 species of fungi representing 71 genera were isolated from litter
samples collected at sites containing healthy American Beech, Fraser Fir,
and Eastern Hemlock during the study (Appendix 1). A total of 3360 fungi
were isolated in 2005 and 3406 in 2006. Of those totals, 38.3% were from
American Beech, 30.6% from Fraser Fir, and 31.1% from Eastern Hemlock.
More than 90% were members of the Deuteromycota (= Fungi Imperfecti),
2.6% members of the Ascomycota, 1.4% members of the Zygomycota, and
6% were representatives of other groups or unknowns.
The most common species identified during the study were Trichoderma
harzianum Rifai, Trichoderma virens (J. Miller et al.) Arx, Virgaria
nigra (Link) Nees, Trichoderma koningii Oudem., Trichoderma hamatum
(Bonord.) Bainier, and Penicillium oxalicum Currie & Thom, all of
which belong to the Deuteromycota. Phymatotrichum omnivorum Duggar
was common on American Beech and Fraser Fir, but only in 2005. Many
isolates were unknown, since the majority did not sporulate in culture.
2009 R.E. Baird, S. Woolfolk, and C.E. Watson 615
Yeasts were isolated at low frequencies from the litter of all three tree
species during the study.
Overall species richness values for the fungi were significantly different
on two selective media across tree species and years (Table 2). Values from
both selective media were greater from American Beech than from Eastern
Hemlock and lowest from Fraser Fir. The DRBC medium had numerically
higher values for all three tree species compared to CYCL. Values were
highest (n = 85) for American Beech litter values using DRBC and lowest
(n = 44) on CYCL from Fraser Fir litter. Total taxa were the same as
values from DRBC, indicating that this medium had the broadest spectrum
for isolation of fungi from litter tissues. Species diversity was also signifi-
cantly greater from American Beech than other tree species, but numerically
greater from Eastern Hemlock than from Fraser Fir (Table 2).
Further analyses of species richness and diversity values were determined
between and among tree species compared to sampling dates for 2005
and 2006 (Figs. 1–4). For American Beech, the number of taxa in May 2006
was greater than for any other sampling dates and tree species (Fig. 1). Furthermore,
values for American Beech were greater for all dates in both years
except that Eastern Hemlock had numerically greater numbers of taxa at 31
compared to 27 for American Beech during July 2006. Species richness values
by sampling date were similar between Fraser Fir and Eastern Hemlock
litter except for September 2005, when Fraser Fir (n = 30) had almost twice
the species present as Eastern Hemlock (n = 16). In 2006, Eastern Hemlock
had significantly greater values in May (n = 41) and July (n = 31) than
Fraser Fir, but were similar during July and September. In 2005, American
Beech had numerically greater species richness values in May than the other
two sampling dates, but for May 2006, the value (n = 45) was significantly
greater than July (n = 27) or September (n = 20). Significant values for Fraser
Fir and Eastern Hemlock occurred during the different sampling dates, but
no trends were observed.
No consistent trends in species diversity were observed between sampling
dates for specific tree species and between tree species (Figs. 2 and 4).
Table 2. Species richness (n) and diversity (H') of fungi isolated from litter of three tree species
from Great Smoky Mountains National Park across two years.
Tree species CYCL DRBC Total taxa Species diversity
American Beech 68aB 85a 86a 3.4a
Eastern Hemlock 49b 62b 63b 2.9b
Fraser Fir 44b 57b 60b 2.8b
LSD (P ≤ 0.05) 7.0 8.0 6.0 0.3
ACYCL: Malt extract – yeast extract – cyclosporine, and DRBC: Dichloran – rose bengal – chloramphenicol
BMeans within columns followed by same letter are not significantly different at the 0.05
616 Southeastern Naturalist Vol. 8, No. 4
Figure 1. Species richness for litter-associated fungi compared among three tree species
by sampling date from the Great Smoky Mountains National Park. Means within
a sampling date and year and followed by same letter are not significantly different
at the 0.05 level. The LSDs were 5.0, 6.0, 4.0 and 6.0, 9.0, 5.0 for May, July, and
September in 2005 and 2006, respectively.
Figure 2. Species diversity of litter-associated fungi of three tree species from the
Great Smoky Mountains National Park. Means within a sampling date and year and
followed by same letter are not significantly different at the 0.05 level. The LSDs
were 0.69, 0.41, 0.48 and 0.31, 0.90, 0.62 for May, July, and September in 2005 and
2009 R.E. Baird, S. Woolfolk, and C.E. Watson 617
Figure 4. Species diversity of litter-associated fungi of three tree species from the
Great Smoky Mountains National Park. Means within a tree species and year and
followed by same letter are not significantly different at the 0.05 level. The LSDs
were 0.60, 0.35, 0.63 and 0.83, 0.54, 0.56 for May, July, and September in 2005 and
Figure 3. Species richness for litter-associated fungi of three tree species compared
by sampling date from the Great Smoky Mountains National Park. Means within a
tree species and year and followed by same letter are not significantly different at the
0.05 level. The LSDs were 7.0, 4.0, 4.0 and 7.0, 6.0, 7.0 for May, July, and September
in 2005 and 2006, respectively.
618 Southeastern Naturalist Vol. 8, No. 4
For example, values for American Beech were significantly greater than for
Fraser Fir and Eastern Hemlock over all three sampling dates in 2005, but
values were similar in 2006. Furthermore, when species diversity was compared
in 2005 or 2006 by sampling dates for each tree species, significant
differences did occur but no consistent trends could be determined (Fig. 4).
Species richness, diversity, and evenness values were also calculated
across pooled sampling date data for the taxa isolated (Table 3). Species
richness values had similar trends as species diversity between sampling
dates and across years. Species richness and diversity were higher in May
than July and September. When total taxa were compared between years,
results varied with no consistent trends. The values for fungi in 2006 were
highest in May (n = 69; H' = 3.2) and lowest in September (n = 24; H' = 2.0),
but evenness varied. Total species richness values also showed a significantly
decreasing trend over time. May had a greater number of taxa (89) than July
(71) and September (58). In addition, significantly greater species diversity
occurred for May litter during both years, compared to July and September.
Overall, litter samples from May 2006 had significantly greater species
richness, diversity, and evenness values than 2005 (Table 3). Furthermore,
no additional significant trends were noted for July or September. The high
isolation frequencies for species such as T. harzianum, T. koningii, and T.
virens contributed to the lower values of evenness for both years of the study
(Appendix 1). Evenness values for fungi pooled across both years, three tree
species, and sampling dates had moderate to high relative abundance at E =
0.66 (data not shown). Evenness values for fungi ranged from E = 0.63 to
0.68 and were similar between litter samples of three tree species, indicating
that the fungal community was generally uniform between the three tree species.
Furthermore, when evenness was compared by sampling date per year
(Table 3), relative high abundance (0.62–0.77) indicates moderate to high
similarity between numbers of individuals isolated per taxa for each year.
When evenness values were compared between years, only May 2006 had a
significantly greater value than the 2005 results.
Coefficient of community values were obtained for fungal taxa by comparing
litter data between the sampling dates and pooled tree species data
(Table 4). Results indicated that almost 50% or greater of the common fungal
Table 3. Species richness (n), diversity (H'), and evenness (E) for fungi identified from litter
of American Beech, Eastern Hemlock, and Fraser Fir from Great Smoky Mountains National
Park over two years. *Indicates significantly different between years for a sampling date using
Jackknifing procedure (P ≤ 0.05). Means within columns followed by same letter are not
significantly different at the 0.05 level.
Richness (n) Diversity (H') Evenness (E)
Sample date 2005 2006 Total taxa 2005 2006 Total taxa 2005 2006 Total taxa
May 54a* 69a* 89a 2.7a* 3.2a* 3.3a 0.68b* 0.77a* 0.66a
July 52a 41b 71b 2.4a 2.7a 2.8b 0.62b 0.73ab 0.63a
September 50a* 29c* 58c 2.8a* 2.0b* 2.7b 0.72a 0.63b 0.68a
LSD 7 9 8 0.34 0.57 0.3 0.07 0.12 0.19
2009 R.E. Baird, S. Woolfolk, and C.E. Watson 619
taxa could be isolated throughout the growing season and for the total years
from the litter of the three tree species. Tree species data pooled across sampling
dates ranged from 0.48 to 0.55 in 2005 and 0.56 to 0.65 in 2006. When
CC values were determined for total years, ≈60% of taxa were common over
the three sampling dates. Furthermore, when comparisons between tree species
were analyzed by year and total years, values were similar with high
levels of similarity (Table 5). Values from 2005 compared to 2006 showed
reverse trends for each tree species litter comparisons. In 2006, CC values
were relatively similar regardless of tree species comparisons.
Percent nutrient composition of the litter samples were analyzed for
ash, organic matter, crude protein, C, N, C:N ratio, and pH. These data
were pooled across years since no significant interaction occurred between
years. When nutrient composition of the litter samples were compared by
sampling dates and between the three tree species, % ash, % crude protein,
and pH were significantly lowest for Fraser Fir than for the other tree species
(Table 6). Also, % N was significantly lower from Fraser Fir litter than
the litter of the other tree species, but highest for % C. In contrast to those
results, C:N ratio for Fraser Fir litter was similar to that of the other two species
analyzed. Also, % organic matter was significantly greater for Fraser Fir
(95.3) than for American Beech (88.6) and Eastern Hemlock (80.0).
Table 6. Percent nutrient contents and soil pH from litter samples of three tree species in Great
Smoky Mountains National Park.
Organic Crude C:N
Tree species Ash matter protein Nitrogen Carbon ratio pH
American Beech 11.4bA 88.6b 11.8a 2.1a 48.8b 23.7b 4.5a
Hemlock 20.0a 80.0c 11.8a 2.2a 49.3b 25.8ab 4.5a
Fraser Fir 4.8c 95.3a 9.6b 1.9b 53.2a 27.0a 3.8b
LSD (P ≤ 0.05) 6 6 0.83 0.2 1.98 2.78 0.17
AMeans within columns followed by same letter are not significantly different at the 0.05 level
using analysis of variance.
Table 5. Coefficient of community values for fungi collected in Great Smoky Mountains National
Litter 2005 2006 Total years
American Beech-Fraser Fir 0.62 0.65 0.70
American Beech-Eastern Hemlock 0.39 0.65 0.62
Fraser Fir-Eastern Hemlock 0.53 0.70 0.70
Table 4. Coefficient of community values for fungi from litter of healthy American Beech,
Fraser Fir, and Eastern Hemlock collected from Great Smoky Mountains National Park.
Years May–July July–Sept. May–Sept.
2005 0.48 0.49 0.55
2006 0.56 0.65 0.65
Total years 0.59 0.57 0.60
620 Southeastern Naturalist Vol. 8, No. 4
For the purposes of this study, litter that was collected under a specific
tree canopy is referred to as belonging to that species (e.g., American Beech
litter). It is important to note that litter samples collected beneath a tree
species really represents a compilation of debris from the immediate plant
community that would directly infl uence fungal diversity. Also, fungal diversity
would be directly infl uenced by the local living plant communities
that occur in direct proximity underneath the same forest tree canopy zones.
However, no plant data was obtained in this study for those comparisons to
The litter study is a continuation of an ongoing ATBI project in GSMNP
to determine the mycobiota associated with organic substrates of American
Beech, Fraser Fir, and Eastern Hemlock. Over 100 species of fungi were
identified, including 55 new records in the Park, during the two-year study.
Previously, bark from the same 3 tree species was assayed over 2 years with
greater than 94 species collected, representing a diverse assemblage of fungi
(Baird et al. 2007).
The major assemblage of fungi isolated in the current litter study conforms
to results obtained from previous research the mycobiota of bark and litter,
with the majority of taxa belonging to the Deuteromycetes (Baird 1991, Baird
et al. 2007, Bills and Polishook 1992, Garg and Sharma 1985). In a study
evaluating bark tissues, the most common genus, Trichoderma, consisted
of 13 species (Baird et al. 2007). Approximately 65% of species identified
from litter were different from the bark study, but we had similar common
fungal species and isolation frequency levels of Trichoderma spp., including
13 species in the current investigation. These fungi are considered important
cellulose-degrading microorganisms that rapidly colonize dying and dead
plant tissues until depletion of the nutrients (Harmon 2000). Trichoderma spp.
are also known to be antagonistic or biological control agents of bacteria and
other fungi (Chaverri and Samuels 2003). A thorough discussion of Trichoderma
on plant tissues and their role in forest ecosystems is available in Baird et
al. (2007). Bills and Polishook (1994) reported that 300 to 400 species of fungi
could be obtained from 1-ml leaf-litter samples collected from the tropics,
which suggests greater species richness in warmer climates. In another study,
48 fungal taxa were identified from Tilia cordata Mill. (Littleleaf Linden) litter,
with the most common species being Cladosporium herbarum (Pers.:Fr.)
Link and Alternaria alternate Keissler (Orazona et al. 2003); only two species
of Trichoderma were identified.
Virgaria nigra (Link) Nees was commonly isolated from the litter of the
three tree species in the current study, and this species had the third highest
isolation frequencies of any species observed during the two-year study. This
fungal species is reported to occur primarily as a saprophyte (Barron 1972).
In the previous ATBI bark tissue study by Baird et al. (2007), other common
fungal taxa following Trichoderma spp. were Curvularia lunata (Wakker)
Boedijn, two Pestalotiopsis spp., and five Penicillium spp.
2009 R.E. Baird, S. Woolfolk, and C.E. Watson 621
In this study, the isolation medium used influenced species richness
levels (Table 2). DRBC had consistently greater isolation frequencies and
species richness than CYCL throughout the study. It has been reported
that the addition of colony-restricting agents such as rose bengal, Dichloran,
or cyclosporine A, could negatively affect growth and sporulation
of different fungal species (Collado et al. 2007). However, these growth
agents are known for minimizing the impact of fast-growing fungi that
prevent overgrowth of taxa that are slow-growing. Many different growth
media were previously used for environmental sampling, but DRBC and
CYCL were reported to allow greater diversity of fungal species (Bills
and Polishook 1994, Bills et al. 2004).
As stated previously, total species richness values for fungi pooled
across sampling dates and years were significantly greater from American
Beech litter than Eastern Hemlock and Fraser Fir litters. When those data
were compared by sampling dates, species richness values were also highest
from American Beech litter, with few exceptions, both years of the study.
Contrary to these results, values were similar between tree species during
the bark study (Baird et al. 2007). Species richness values from litter by year
varied between sampling dates without any definitive trends. As observed in
this study, forest litter has a diverse microbial community similar to what has
been shown for soils (Barron 1972, Orazona et al. 2003).
Total species diversity showed a trend similar to that observed as species
richness for fungi on American Beech and other tree species (Table 2). Diversity
of taxa was significantly greater for American Beech across years than for
Fraser Fir and Eastern Hemlock. When values were compared by sampling
dates and years, American Beech generally had the greatest diversity except
for September of both years (Fig. 2), where Fraser Fir had numerically greater
values. Furthermore, species diversity compared by sampling dates for each
tree species or pooled across species varied with no apparent trends noted (Table
4, Fig. 4). Fraser Fir diversity had a reverse trend when compared between
sampling dates for each year. Factors previously stated such as select nutrients
and pH levels may have been important soil and environmental conditions affecting
diversity values. Also, no apparent trends from CC values were noted
by sampling date or between litter types.
Litter provides a complex nutrient base enabling a diverse assemblage of
fungi to colonize plant tissues during various stages of biodegradation (Ingham
et al. 1985, Polishook 1996). The pH levels of forest litter were similar
between American Beech and Eastern Hemlock, but lowest for Fraser Fir.
The lower pH of the Fraser Fir litter may be responsible, at least in part, for
lower species richness, since hydrogen-ion concentrations of the samples
affect growth of fungi (Lilly and Barnett 1951). In a previous study, myxomycete
richness and diversity compared on various plant substrates were
affected by pH levels within those tissues (Stephenson 1989, Stephenson
et al. 2004). Conifers generally require more acid conditions for growth,
thus limiting species richness. Another chemical property assayed was ash
622 Southeastern Naturalist Vol. 8, No. 4
content of the litter samples. Fraser Fir litter had significantly lower (4.8%)
ash content than Eastern Hemlock (20.0%) and American Beech (11.4%) litters.
Ash, which consists of inorganic minerals such as silicon (Si), calcium
(Ca), potassium (K), sulfur (S), and clorine (Cl) (Bakker and Elbersen 2005),
are important in survival and reproduction in different fungi (Lilly and Barnett
1951). These results indicate that percent ash levels in Fraser Fir litter
may have been another limiting factor in species richness values from these
Elevation differences were reported to have an impact on forest soil C
and N dynamics in GSMNP by Garten and Van Miegroet (1994). Results
from that study showed that ecosystem N increases with elevation in the
park. In this study, the litter nutrient levels of N were numerically lower
(1.9%) for Fraser Fir litter than for American Beech (2.1%) and Eastern
Hemlock (2.2%) litters. Collection site elevations for Fraser Fir were greater
than for American Beech, and N was significantly lower for Fraser Fir litter.
In addition, C was significantly greater in Fraser Fir litter than for American
Beech and Eastern Hemlock litters. When C:N ratio was compared, Eastern
Hemlock litter had a significantly higher ratio (27.0%) than American Beech
litter (23.7%), but Fraser Fir litter (25.8%) was similar to both tree species.
The C:N ratio balance has been shown to be important for successful growth
and reproduction of fungi, and most taxa generally require ≈30:1 for vigorous
growth (Barron 2003).
Organic matter content, which is a source of polysaccharides, lipids, nucleic
acids, and proteins, was significantly higher for Fraser Fir litter (95.3%)
than American Beech litter (88.6%) and Eastern Hemlock litter (80.0%).
These components in organic matter are generally unavailable and must be
degraded before fungal utilization (Barron 2003). These latter results may
be a good indicator that organic matter degradation is slower in Fraser Fir
forests, thus making nutrients less available at the higher elevations. Stephenson
et al. (2004) reported that a similar trend of decreasing numbers
of species of myxomycete occurred with increased elevation. Potentially,
differences in elevations, acidic nature of the soils, and other factors affect
a microorganism’s ability to grow and process the organic matter. Another
limiting factor in low organic matter degradation is that Fraser Fir stands are
located in boreal climatic zones in the park. Shanks’ (1954) climate study of
the GSMNP reported that growing-season temperatures above 1500 m elevation
in the spruce-fir zone averaged 5.5 °C to 8.3 °C lower than at the base
of the mountain. Average annual temperatures in Fraser Fir forests can range
from 7.5 °C at an altitude of 1920 m in the GSMNP (Shanks 1954) to 6 °C
at an altitude of 2037 m on Mount Mitchell in the Black Mountains (Oosting
and Billings 1951). In a previous study, organic-matter decomposition was
observed to decline with increasing elevation due to colder temperatures
(Garten and Van Miegroet 1994). It was reported that 50% higher precipitation
occurs at the higher elevations of the spruce-fir zone (Shank 1954).
Furthermore, annual precipitation is ≈147 cm at low elevations and ≈222 cm
2009 R.E. Baird, S. Woolfolk, and C.E. Watson 623
at the high elevations, but temperature is the critical factor in organic-matter
degradation (Garten and Van Miegroet 1994).
Results from this litter study and the previous bark-sampling project
(Baird et al. 2007) further confirmed that the Deuteromycota are the most
prevalent group of fungi that occur across the three tree species. Most of
these anamorphic species have been shown to be members of the Ascomycota.
Significantly greater species richness levels were recorded for
American Beech litter than for the other two tree species. In addition, over
half the taxa present on litter tissues differed from those isolated from bark
samples in the previous ATBI study by Baird et al. (2007). Overall, species
richness, diversity, and evenness data, when compared separately or pooled
across tree species, sampling dates, and years, showed no apparent trends. It
was further observed that environmental and site parameters, such as elevation,
temperature, pH, or chemical composition of litter tissues, may have
affected species richness, diversity, and evenness. Even though the results
from cultural media yielded over 100 fungal species, growth inhibitors in
the media may have limited the diversity. Therefore, studies using molecular
techniques are now underway, evaluating litter and bark samples of these
tree species to determine if a more diverse group of fungi can be identified
from those same tissues types.
I would like to acknowledge Discover Life in America for providing research support
during the second year of the study under Project Number GSM #2006-01. Also,
thanks are extended to Emily Tuck for laboratory support during the investigation and
to Robyn Hearn, MSU, for graphics support.
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2009 R.E. Baird, S. Woolfolk, and C.E. Watson 627
Appendix 1. Mean percent occurrence of fungi from ground litter of three tree species
from Great Smoky Mountains National Park. Percent isolation frequency based
on 3 sampling dates x 4 replicate trees x 2 media x 10 plates/medium x 5 isolates/
plate= 1200/ tree species; sometimes less than 5 isolates/plate were found. A.B. =
American Beech, F.F. = Fraser Fir, and E.H. = Eastern Hemlock.
Taxa A.B. F.F. E.H. A.B. F.F. E.H.
Acremonium crotocinigenum <1.0 0.0 0.0 <1.0 <1.0 3.3
(Schol-Schwarz) W. Gams
A. hansfordii (Deighton) W. Gams 0.0 0.0 <1.0 0.0 0.0 0.0
Acremonium spp. Link: Fr. <1.0 <1.0 0.0 <1.0 0.0 <1.0
Acladium conspersum Link: Fr. 0.0 0.0 0.0 <1.0 0.0 <1.0
Alternaria tenuis Nees <1.0 0.0 0.0 0.0 0.0 0.0
Amblysporium spongiosum (Pers.) S. Hughes <1.0 <1.0 0.0 0.0 0.0 <1.0
Aspergillus niger Tiegh. 0.0 0.0 0.0 0.0 0.0 <1.0
Aspergillus spp. <1.0 <1.0 <1.0 0.0 0.0 0.0
Aposphaeria pezizoides Ellis & E. <1.0 0.0 0.0 <1.0 0.0 0.0
Aureobasidium pullulans (deBary) G. Arnaud <1.0 <1.0 0.0 <1.0 <1.0 <1.0
Bactodesmium obliquum Sutton <1.0 0.0 0.0 0.0 0.0 <1.0
Bipolaris sorokinianum (Sacc.) Shoemaker <1.0 0.0 0.0 0.0 0.0 0.0
Candida guilliermondii (Castellani) <1.0 0.0 0.0 0.0 <1.0 0.0
Langeron & Guerra
Candida spp. Berkhout <1.0 1.9 0.0 <1.0 <1.0 0.0
Cephalosporium sp. Corda <1.0 1.4 <1.0 <1.0 0.0 0.0
Chaetomella oblonga Fuckel 0.0 0.0 <1.0 0.0 0.0 0.0
Chaetopsina fulva Rambelli <1.0 <1.0 0.0 0.0 0.0 0.0
Chloridium chlamydosporum (Beyma) <1.0 0.0 0.0 0.0 0.0 0.0
Cladosporium herbarum (Pers.: Fr.) Link <1.0 0.0 0.0 <1.0 0.0 0.0
Cladosporium spp. Link <1.0 <1.0 0.0 <1.0 0.0 <1.0
Curvularia oryzae Bugnicourt <1.0 0.0 0.0 0.0 0.0 0.0
Dactylella leptospora Drechsler 0.0 0.0 0.0 <1.0 <1.0 0.0
Diheterospora chlamydosporia (Goddard) <1.0 0.0 0.0 0.0 0.0 0.0
Barron & Onions
Fusarium equiseti (Corda) Sacc. 0.0 0.0 0.0 0.0 <1.0 0.0
F. lateritium Nees.: Fr. <1.0 0.0 0.0 0.0 0.0 1.1
F. nivale (Fr.) Ces. 1.2 0.0 <1.0 <1.0 0.0 0.0
F. oxysporum Schlechtend.: Fr. 0.0 <1.0 0.0 0.0 0.0 0.0
F. sambucinum Fuckel 0.0 1.0 <1.0 0.0 0.0 0.0
F. semitectum Berk. & Ravenel <1.0 <1.0 0.0 0.0 0.0 0.0
F. solani (Mart.) Sacc. <1.0 <1.0 0.0 0.0 0.0 0.0
F. verticillioides (Sacc.) Nirenberg <1.0 0.0 0.0 0.0 0.0 0.0
Fusarium spp. Link:Fr. <1.0 <1.0 0.0 <1.0 <1.0 <1.0
628 Southeastern Naturalist Vol. 8, No. 4
Taxa A.B. F.F. E.H. A.B. F.F. E.H.
Geotrichum candidum Link <1.0 0.0 <1.0 0.0 0.0 0.0
Gliocladium deliquescens Sopp. 0.0 0.0 0.0 <1.0 0.0 0.0
Gliomastix murorum (Corda) Hughes 0.0 <1.0 0.0 0.0 0.0 0.0
Gonotobotryum apiculatum (Peck) Hughes <1.0 0.0 0.0 0.0 0.0 0.0
Hemicorynespora deightonii M.B. Ellis <1.0 0.0 0.0 0.0 0.0 0.0
Humicola sp. Traaen <1.0 <1.0 0.0 <1.0 <1.0 0.0
Idriella sp. Nelson & Wilhelm 0.0 0.0 0.0 <1.0 0.0 <1.0
Ingoldia craginiformis Petersen 0.0 0.0 <1.0 0.0 0.0 0.0
Leptostroma caricinum Fr.:Fr. <1.0 0.0 0.0 0.0 0.0 0.0
Memnoniella echinata (Rivolta) Galloway Sm. <1.0 0.0 0.0 <1.0 0.0 0.0
Menispora glauca Pers. <1.0 0.0 0.0 0.0 0.0 0.0
Monascus spp. Zukal 0.0 0.0 0.0 0.0 <1.0 0.0
Monochoetia concentrica (Berk. & Broom) <1.0 0.0 0.0 <1.0 0.0 0.0
Sacc. & Sacc.
Monochaetia sp. (Sacc.) Allesch. 0.0 0.0 0.0 <1.0 0.0 <1.0
Monocillium indicum Saksena 0.0 <1.0 <1.0 0.0 0.0 0.0
Nigrospora sphaerica (Sacc.) E. Mason 0.0 <1.0 0.0 0.0 0.0 0.0
Nodulosporium spp. G. Preuss 0.0 1.0 0.0 0.0 0.0 0.0
Oidiodendron griseum Robak <1.0 0.0 0.0 0.0 0.0 0.0
Paecilomyces fumosoroseus (Wize) A.H.R. <1.0 1.2 0.0 0.0 <1.0 <1.0
Brown & G. Smith
Paecilomyces spp. Bainier 1.8 2.1 <1.0 <1.0 0.0 <1.0
Penicillium arenicola Chalabuda 0.0 <1.0 0.0 <1.0 1.8 1.2
P. islandicum Sopp. <1.0 0.0 0.0 <1.0 <1.0 1.5
P. lividum Westling <1.0 <1.0 0.0 1.1 1.0 <1.0
P. oxalicum Currie & Thom 1.3 2.3 2.4 1.6 1.0 8.3
P. sclerotiorum Beyma <1.0 0.0 0.0 <1.0 <1.0 <1.0
Penicillium spp. Link:Fr. 1.2 1.9 1.0 1.2 2.6 7.0
Periconia macrospinosa Lefebvre and 0.0 0.0 0.0 <1.0 0.0 0.0
Pestalotia clavispora Atk. 1.2 1.8 <1.0 <1.0 <1.0 3.6
Pestalotiopsis guepini (Desm.) Steyaert 7.3 <1.0 <1.0 <1.0 <1.0 <1.0
Phialophora verrucosa Medlar <1.0 0.0 0.0 <1.0 0.0 0.0
Phoma dura Sacc. <1.0 <1.0 0.0 <1.0 0.0 0.0
Phymatotrichum omnivorum Duggar 1.1 9.8 0.0 0.0 0.0 0.0
Pithomyces atro-olivaceous <1.0 0.0 0.0 <1.0 0.0 0.0
(Cooke & Harkin.) M.B. Ellis
Rhinocladiella atrovirens Nannf. 0.0 0.0 0.0 <1.0 0.0 0.0
Rhizoctonia solani Kühn (AG-3) 0.0 <1.0 0.0 0.0 0.0 0.0
Rhizosphaera pini (Corda) Maubl. <1.0 <1.0 0.0 0.0 0.0 <1.0
Rhynchophoma sp. P. Karst. 0.0 0.0 <1.0 0.0 0.0 0.0
Sarocladium sp. Gams & Hawksworth 0.0 0.0 <1.0 0.0 0.0 <1.0
Seiridium sp. A. Nees <1.0 0.0 0.0 0.0 0.0 0.0
Seiridium sp. B. Nees <1.0 0.0 0.0 0.0 0.0 0.0
2009 R.E. Baird, S. Woolfolk, and C.E. Watson 629
Taxa A.B. F.F. E.H. A.B. F.F. E.H.
Sporothrix schenckii Hektoen & Perkins <1.0 0.0 0.0 0.0 0.0 0.0
Stilbum sp. Tode 0.0 <1.0 <1.0 0.0 0.0 0.0
Thysanophora canadensis Stolk & Hennebert 0.0 0.0 0.0 0.0 0.0 <1.0
Trichoderma aggressivum Samuels & W. Gams 0.0 <1.0 0.0 4.6 0.0 0.0
T. atroviride P. Karst. 0.0 0.0 0.0 <1.0 <1.0 <1.0
T. aureoviride Rifai <1.0 0.0 8.2 <1.0 <1.0 <1.0
T. cremeum Chaverri & Samuels 0.0 0.0 0.0 <1.0 <1.0 <1.0
T. ghanense Y. Doi, Y. Abe & J. Sugiyama 0.0 0.0 0.0 5.4 0.0 <1.0
T. hamatum (Bonord.) Bainier 0.0 <1.0 1.7 15.2 8.0 3.5
T. harzianum Rifai 15.9 15.5 27.7 16.9 16.8 17.3
T. koningii Oudem. 6.4 8.0 8.9 1.6 <1.0 4.1
T. stromaticum Samuels & Pardo – Schulth. 0.0 0.0 0.0 0.0 <1.0 <1.0
T. virens (J. Miller et al.) Arx 5.4 2.4 8.8 8.4 16.2 9.1
T. viride Pers.:Fr. 3.9 1.1 0.0 12.3 <1.0 1.3
Trichoderma spp. Pers. 3.0 2.4 4.4 <1.0 <1.0 <1.0
Trichosporiella cerebriformis 0.0 0.0 0.0 <1.0 0.0 0.0
(G.A. de Vries & Kleine-Natrop) W. Gams
Trichosporon sp. Behrend <1.0 0.0 0.0 0.0 0.0 0.0
Truncatella angustata (Pers.) S. Hughes <1.0 0.0 0.0 <1.0 0.0 0.0
Ulocladium spp. Preuss <1.0 0.0 0.0 0.0 <1.0 0.0
Verticillium sp. Nees <1.0 <1.0 0.0 <1.0 0.0 1.0
Virgaria nigra (Link) Nees 0.0 0.0 0.0 0.0 0.0 <1.0
Unknown spp. 9.0 6.7 2.6 5.3 8.8 3.5
Unknown spp. 0.0 0.0 0.0 <1.0 <1.0 <1.0
Eupenicillium cinnamopurpurem 1.1 0.0 <1.0 1.5 <1.0 <1.0
D.B. Scott & Stolk
Eurotium amstelodami L. Mangin 0.0 0.0 0.0 0.0 0.0 <1.0
Eurotium rubrum W. Bremer 0.0 0.0 0.0 <1.0 0.0 <1.0
Sordaria sp. Ces. & De Not. 0.0 <1.0 2.5 <1.0 0.0 <1.0
Unknown spp. <1.0 <1.0 0.0 0.0 0.0 0.0
Absidia spp. Tiegh. <1.0 0.0 <1.0 <1.0 <1.0 <1.0
Mortierella sp. Coem. 0.0 0.0 1.6 1.8 <1.0 <1.0
Mucor microsporus Naumov. <1.0 <1.0 0.0 0.0 0.0 <1.0
Rhizopus arrhizus A. Fisher 0.0 0.0 0.0 0.0 0.0 <1.0
R. monosporus Tiegh. 0.0 0.0 0.0 0.0 0.0 <1.0
R. niger (Ciagl. & Hewelke) Gedoelst 0.0 0.0 0.0 0.0 0.0 <1.0
630 Southeastern Naturalist Vol. 8, No. 4
Taxa A.B. F.F. E.H. A.B. F.F. E.H.
Rhizopus stolonifer (Ehrenb.:Fr.) Vuill. 0.0 0.0 0.0 0.0 0.0 <1.0
Rhizopus spp. Ehrenb. 0.0 0.0 0.0 0.0 0.0 <1.0
Rhopalomyces elegans Corda <1.0 0.0 0.0 0.0 0.0 0.0
Phytophthora spp. deBary <1.0 0.0 0.0 0.0 0.0 0.0
Pythium spp. Pringsh. 1.0 <1.0 0.0 <1.0 0.0 0.0