Mycologia, 98(2), 2006, pp. 353–363.
# 2006 by The Mycological Society of America, Lawrence, KS 66044-8897
A microtiter plate procedure for evaluating fungal functional diversity on
nitrogen substrates
Heath W. Grizzle1
John C. Zak2
Department of Biological Sciences, Texas Tech
University, Mailstop 3131, Lubbock, Texas 79401
Abstract: Ascertaining the effects of anthropogenic
disturbance on belowground diversity is of para-
mount importance because pollution from agricul-
tural practices and industrialization are increasing
worldwide. Although we have methods for evaluating
soil microbial function with respect to carbon use our
ability to evaluate use of other compounds is limited.
Because N cycling is of paramount importance in
ecosystem stability, evaluation of the ability of
saprophytic soil fungi to use a variety of N sources
would provide important information on possible
alterations in ecosystem stability with disturbance.
Herein is described a procedure (soil Nitrolog) for
evaluating fungal functional diversity on a suite of 95
different N substrates. The soil Nitrolog procedure
was evaluated by testing fungal functional diversity at
two sites in Big Bend National Park (Chihuahuan
Desert), differing in elevation and plant community
composition. The soil Nitrolog procedure distin-
guished between the two sites based on overall use
of the 95 N substrates. In addition the procedure
detected differences in individual substrate use based
on site specific plant compounds in response to
changes in the amount of N entering these ecosys-
tems from anthropogenic inputs.
Key words: Big Bend National Park, Biolog,
Chihuahuan Desert, functional diversity, soil fungi,
soil nitrogen
INTRODUCTION
Decomposition is as vital to ecosystem function as is
primary production (Parkinson and Coleman 1991).
Therefore changes in decomposer community struc-
ture and activities as a consequence of disturbance,
climate change or anthropogenic inputs can have
important repercussions on ecosystem functioning
(Perry et al 1989). As the primary decomposers of
litter and soil organic matter in terrestrial ecosystems
soil fungi are vital for the maintenance of bio-
Accepted for publication 20 Jan 2006.
1
Corresponding author. E-mail: hgrizzle@gmail.com
2
E-mail: john.zak@ttu.edu.
353
geochemical cycling. Biogeochemical cycling returns
nutrients to the soil solution for uptake by plants,
bacteria and other fungi (Cook and Rayner 1984).
Kandeler (1996) noted that, although carbon cycling
may not be altered by changes in community
composition, nitrogen dynamics are less resilient.
Because the supply of nutrients is important ‘‘bottom
up’’ controls of ecosystem structure and dynamics
( Jenny 1980) a disturbance altering resource avail-
ability would affect other ecosystem processes (Cha-
pin et al 1997). Understanding how saprophytic soil
fungi control decomposition rates and participate in
seasonal patterns of nutrient availability may prove
crucial in predicting immediate and lasting effects of
multiple stressors, such as climate change and
anthropogenic disturbances on ecosystem process
and stability. Providing a community level assessment
of the ability of soil fungi to catabolize an array of
nitrogenous compounds is critical to linking the
functional diversity of soil fungi and effects of
disturbance or environmental stressors.
Effects of atmospheric N deposition on saprophytic
soil fungal biodiversity and activity has received little
attention with most of the research focused on
changes to mycorrhizal taxa and patterns of fruit
body production (Hawksworth and Colwell 1992,
Vogt et al 1992). Our understanding of the interac-
tions among soil N dynamics, atmospheric N inputs
and saprophytic soil fungi is limited primarily to
fertilization effects in agricultural systems (Donnison
et al 2000). In one of the few studies of N effects on
soil saprophytic fungi in nonagricultural systems
Arnebrant et al (1990) observed a direct decline in
decomposer soil fungi in response to ammonium
nitrate or urea applications to a Scots pine forest. This
result is mirrored in studies from agricultural systems
that also report changes in fungal biodiversity and
a shift to a bacterial dominated system with increased
N inputs (Bardgett and Leemans 1995, Bardgett et al
1996). More recently Bardgett et al (1999) suggest
that in grassland ecosystems, as N levels increase, the
role of soil fungi in decomposition and nutrient
cycling decreases substantially. Changes that occur in
soil fungal species composition and functional di-
versity in response to altered precipitation patterns,
coupled with anthropogenic inputs of N, should have
lasting effects on ecosystem functioning, plant success
and successional trajectories. Ecosystem response to
concurrent changes in below- and aboveground
354
processes and attendant fungal biodiversity is com-
plex and difficult to predict. Such interactions
include effects on food web processes and antagonis-
tic, symbiotic and mutualistic relationships among
plants and soil microbes (Wolters et al 2000).
The ability to evaluate fungal functional diversity
with respect to organic and inorganic N use, either
separately or in conjunction with C use, would be
useful in determining differences in the functional
ability of fungi between ecosystems as they are
influenced by vegetation structure, land use, distur-
bance history, agriculture, anthropogenic effects of N
and historical amounts of soil N. The Fungilog
(Dobranic and Zak 1999) and soil Fungilog method
(Sobek and Zak 2003) were introduced as a means of
relating ecosystem processes to the functional di-
versity of an assemblage of litter and soil fungi as
evaluated from the in vitro catabolic profile of the
attendant fungi. Fungal functional diversity as evalu-
ated by these methods is defined as the rate at which
C compounds are used (by substrate activity) and
the numbers of compounds catabolized (affecting
substrate richness) from a group of 95 C com-
pounds by an assemblage of fungi colonizing plant
litter or soil organic matter (Dobranic and Zak 1999,
Sobek and Zak 2003). Previous studies describing
functional diversity in microbial communities have
focused on C use (Garland and Mills 1991, Zak et al
1994, Haack et al 1995, Zhang and Zak 1995, Guckert
et al 1996, Willig et al 1996, Pennanen et al 1998,
Smalla et al 1998, Dobranic and Zak 1999, Sobek and
Zak 2003) to define functional differences among
ecosystems.
Biolog Inc. recently introduced a microtiter plate
(PM3) that allows for the evaluation of inorganic and
organic nitrogen use by strains of bacteria. Described
here is a method for evaluating soil fungal functional
diversity of N substrates (Nitrolog) found on PM3H
microtiter plates. The soil Nitrolog method is based
on the soil Fungilog method described by Sobek and
Zak (2003). The objectives of this study were: (i)
develop a protocol that would let one use the Biolog
PM3 microtiter plates to evaluate the N use capabil-
ities of soil fungi and (ii) evaluate the sensitivity of the
method to detect differences in the ability of soil
fungi to use a range of N compounds between two
elevation zones along an elevational gradient in the
Chihuahuan Desert and in response to nitrogen
additions.
METHODS
Site description.—Data contained herein were col-
lected from two distinct vegetation zones along the
Pine Canyon Watershed (Hermann et al 2000) in
MYCOLOGIA
Big Bend National Park (BBNP). BBNP is located
in southwestern Texas within the Chihuahuan
Desert. BBNP comprises arid and semiarid envir-
onments and is characterized by low annual
precipitation (330 mm), of which the majority
occurs in the summer. Seasonal temperature
fluctuations are characterized by hot summers
and cool winters. The average winter temperature
is 10 C, and the average summer temperature is
27 C (Staff 1985).
Soil samples were collected from the high elevation
oak-pine forest on Lost Mine Peak and the mideleva-
tion sotol grassland. The oak-pine forest (LM,
elevation 2098 m) is the highest point in the Pine
Canyon Watershed and is dominated by Quercus
emoryi (a species of live oak), Pinus cembroides (pinyon
pine), several Juniperus species and several genera of
bunch grasses. The sotol grassland (SG, elevation
1526 m) is a grama grassland characteristic of
midelevation grassland in this part of the Chihuahuan
Desert. These grasslands are dominated by Dasylirion
leiophyllum (sotol), Nolina texana (bear grass), several
species of Opuntia (prickly pear) and Bouteloua
curtipendula (side-oats grama) (Dobranic and Zak
1999). The two study sites are in the northeastern
portion of the Chisos Mountains and are separated by
approximately 550 m elevation and 1.75 km.
The sites used to evaluate the effectiveness of the
Nitrolog procedure in detecting landscape level
patterns in the ability of soil fungi to use nitrogen
compounds are part of a larger effort to understand
the affects of anthropogenic N deposition and
climatic change on soil microbial dynamics within
an arid landscape.
Method development.—BiologH PM3 plates, which
contain 95 different nitrogen compounds, esti-
mate N use in a minimally defined medium and as
such contain no C source. Therefore a C source
must be added to the PM3 plates (Biolog instruc-
tions for PM3 plates). An appropriate C source
and concentration of C needed to be determined
to allow for a gradual reduction of the tetrazolium
dye over time to ensure that slowly growing fungi
would contribute to color development in in-
dividual wells. Glucose was chosen as a standard C
source for addition to the microtiter plates
because it is readily catabolized by soil fungi
(Griffin 1994). A series of glucose concentrations,
0.0 g L21, 0.1 g L21, 1.0 g L21 and 5.0 g L21, were
evaluated to determine the appropriate amount of
glucose to be added with the soil organic matter. A
concentration of glucose would be unacceptable
if: (i) the tetrazolium indicator was maximally
reduced within 24 h after incorporation of fungal
 GRIZZLE AND ZAK: FUNGAL FUNCTIONAL DIVERSITY ON NITROGEN
inoculum to the microtiter plate because slow
growing fungi would not have contributed to the
catabolism in the well; and (ii) concentrations
resulted in an absorbance reading greater than
3.00 in any well before the end of the 5 d
incubation period (the maximum absorbance
reading of the plate reader is 3.0). In addition
the concentration of glucose should provide an
absorbance value that was similar to that observed
for the glucose well in the SFN-2 Biolog plates
used for C evaluation (Sobek and Zak 2003). With
levels of fungal activity similar between the two
types of Biolog microtiter plates using glucose as
the standard, one then could directly compare
activity on the C vs. the N source plates.
For evaluation of the optimal levels of glucose, five
300 g soil samples were collected along two 100 3
30 m belt transects established in the sotol grassland
from Pine Canyon at Big Bend National Park in Jan
2003. Soil samples were sieved (2 mm screen) in the
field to remove stones, roots and other debris. Soils
were stored no more than 1 wk at 4 C before
processing. In the lab soil organic matter particles
(SOMP) were collected from each sample by shaking
approximately 50 g of soil in 100 mL of RO water with
a 10 mL Tween 80 solution for 1 h. This is an essential
step because it removes spores allowing for inocula-
tion of only those fungi actively growing in the soil
(Harley and Waid 1955, Bisset and Widden 1972,
Baath 1988, Sobek and Zak 2003). Each soil sample
subsequently was washed through 500 mm and
250 mm sieves for several minutes with tap water.
SOMP were collected in a beaker from the 250 mm
sieve and filtered through a vacuum filtration system.
SOMP were collected on P8 filter paper (Fisher
Scientific) and stored in a Petri dish at 4 C overnight
and used to inoculate the PM3 plates.
Fifty mg of SOMP, previously determined to be the
density which maximized substrate activity levels but
caused the least OD distortion (Sobek and Zak 2003),
from each soil sample were added to 20 mL of 0.2%
H2O agar in a 25 mL vial that contained 125 mL of
a MTT tetrazolium dye (stock solution is 160 mg of
tetrazolium dissolved in 10 mL of sterile H2O) and
glucose (0 mg, 10 mg, 100 mg and 500 mg/microplate
well respectively) to produce one of the four
concentrations of glucose listed above. Full details
for dissolving the tetrazolium are provided in Sobek
and Zak (2003). Two hundred mL of an antibiotic
solution containing streptomycin sulfate (40 mg) and
chlortetracycline HCL (30 mg/40 mL) are added to
each vial to reduce bacterial contamination (10 mg of
streptomycin and 5 mg of chlortetracycline/micro-
plate well). The H2O agar suspension is vortexed to
evenly distribute the inoculum, tetrazolium dye,
355
glucose and antibiotics. The suspension then is
poured into V-shaped BiologH reservoirs. An eight-
channel micropipetter fitted with large-orifice pipette
tips (1–200 mL capacity) was used to deposit 150 mL
into each well of a BiologH PM3 microtiter plate.
Plates were placed in a Ziploc bag to conserve
moisture, incubated at 25 C and scanned with
a computer-controlled microplate reader at 590 nm
every 24 h for 120 h. Readings were not collected at
96 h due to investigator error.
Method evaluation.—Once an appropriate glucose
concentration was chosen the procedure was field
tested with SOMP collected from the grassland
and oak-pine forest sites in Aug 2003 along the
Pine Canyon Watershed at Big Bend National
Park. Soil samples were collected from a series of
plots at these two locations that were initiated to
examine short-and long-term effects of increasing
atmospheric deposition of N to these desert
ecosystems. In each site an area 40 m 3 33 m
was divided into a grid containing a total of 30, 5 3
5 m plots having 2 m walkways between each plot.
Plots were divided into three treatments with 10
plots randomly assigned to each treatment. Treat-
ments were chosen to evaluate a simulated in-
crease in anthropogenic N deposition. Since 1980
the National Atmospheric Deposition Program
(NADP) has been measuring N deposition from
rainfall in Big Bend National Park. The average N
deposition over this period is 3.99 kg/h/y. The
treatments for each plot are: X, receiving no
additional N, 2X receiving 2.5 g NH4NO3 and
8.24 g CaNO3 (23 annual N deposition), and 4X
receiving 5.0 g NH4NO3 and 16.48 g CaNO3 per
year (43 annual N deposition). Treatments were
applied in Jun and Jul 2003 as half the annual
amount during each month. Soil samples to
a depth of 15 cm were collected in Aug 2003 for
the evaluation component of the study. SOMP
were collected and inoculated into microtiter
plates as described above. Plates were incubated
at 25 C and scanned on the plate reader every
24 h for 5 d.
Analytical approaches.—Functional diversity. Func-
tional diversity was evaluated as substrate activity
(SA, sum of optical densities in all wells/plate),
which is a measure of the rates at which fungal
assemblages can catabolize the various N sub-
strates, and substrate richness (SR, total number
of N substrates catabolized), which is a measure of
the diversity of an fungal assemblage with respect
to the substrates that can be used (Dobranic and
Zak 1999, Sobek and Zak 2003). Guilds of related
compounds (TABLE I) also can be used to evaluate
356 MYCOLOGIA

TABLE I. Nitrogen compounds in guilds found on Biolog PM3 plates

Inorganic N Amines Amides Nucleotides and Nucleosides

Ammonia Hydroxylamine Acetamide Adenine

Nitrate Methylamine Formamide Adenosine
Nitrite N-Amylamine Glucuronamide Cytidine
Urea N-Butylamine D,L-Lactacide Cytosine
Ethylamine Guanine
Ethanolamine Guanosine
Ethylenediamine Thymine
Putrescine Thymidine
Agmatine Uracil
Histamine Uridine
b-phenylethyl-amine Inosine
Tyramine Xanthine
D-Glucosamine Xanthosine
D-Galactosamine Uric Acid
D-Mannosamine Alloxan
N-Acetyl-D-Glucosamine Allantoin
N-Acetyl-D-Galactosamine
N-Acetyl-D-Mannosamine
D-Amino Acids L-Amino Acids Di Amino Acids Fatty Acids
D-Alanine L-Alanine Ala-Asp e-Amino-N-Caproic Acid
D-Asparagine L-Arginine Ala-Gln D,L a-Amino-N-Caprylic Acid
D-Aspartic Acid L-Asparagine Ala-Glu D-Amino-N-Valeric Acid
D-Glutamic Acid L-Aspartic Acid Ala-Gly 2-Amino-N-Valeric Acid
D-Lysine L-Cysteine Ala-His
D-Serine L-Glutamic Acid Ala-Leu
D-Valine L-Glutamine Ala-Thr
Glycine Gly-Asn
L-Histidine Gly-Gln Miscellaneous Substrates
L-Isoleucine Gly-Glu Biuret
L-Leucine Gly-Met N-Acetyl-D-Glutamic Acid
L-Lysine Met-Ala N-Phtyhaloyl-L-Glutamic Acid
L-Methionine L-Pyroglutamic Acid
L-Phenylalanine Parabnic Acid
L-Proline D,L-a-Amino N-Butyric Acid
L-Serine c-Amino-N-Butyric Acid
L-Threonine
L-Tryptophan
L-Tyrosine
L-Valine
L-Citruline
L-Homoserine
L-Ornithine

differences between groups (Zak et al 1994, Willig
et al 1996, Sobek and Zak 2003). Utilization
patterns of individual substrates can be used to
discriminate between sites.
Data analysis. All statistical analyses were per-
formed with SPSS 9.0 (www.spss.com) or Matlab 6.0
(www.mathworks.com). All Matlab programs used
are available at the Texas Tech Biological Sciences
Website (www.biol.ttu.edu/Strauss/Matlab/matlab.
htm). These functions were used: (i) DISCRIM, (ii)
STEPDISK and (iii) PCACORR.
Statistical analysis and site discrimination. SA
and SR. Differences in SA and SR between glucose
concentrations were evaluated with repeated mea-
sures ANOVA. Before conducting each ANOVA,
normality of error terms was evaluated via Kolmo-
gorov-Smirnov test for goodness of fit and homo-
scedasticity was evaluated via Levene’s test for
 GRIZZLE AND ZAK: FUNGAL FUNCTIONAL DIVERSITY ON NITROGEN
TABLE II. Results of Principal Component Analysis for soil
fungi isolated from the Sotol Grasslands and the high
elevation Oak-Pine forest at Big Bend National Park
Upper CI Lower CI
Percent Variance 38.7 37.05 51.92
Explaineda
Component Loadingsb
Site 0.596 20.525 0.987
Treatment 0.695 20.122 0.994
Interaction 0.568 20.394 0.983
a
Percent variance explained by PC1.
b
Component loadings are expressed as correlation coeffi-
cients with PC1.
equality of variances. For the field assessment
differences in overall SA and SR were evaluated
via factorial ANOVA with site and treatment as
factors. A posteriori Fishers LSD tests were per-
formed if ANOVA indicated a significant effect of
date or treatment. Sequential Bonferroni correc-
tions (Sokal and Rohlf 1995) were applied to
control experiment wise error rate.
Individual substrate data. Analysis of substrate
data employed a factorial design with each of the
95 nitrogen substrates being a dependant variable
and site and treatment as factors. To determine
which N substrates maximized separation between
site and N addition and their interaction, a variable
reduction step was necessary. Principal Compo-
nents Analysis (PCA) was chosen to determine
whether a single set of variables maximized group
separation for the combined effects of the factors
and their interaction. PCA requires that the
number of observations be greater than the
number of variables. To meet this assumption
univariate two-way ANOVA were performed on
each of the 95 nitrogen substrates. The resulting F
statistics for site, treatment and interaction were
used as the three variables in the PCA. For each
PCA 5000 bootstrap iterations were performed to
build a sampling distribution against which the
observed data were compared. Confidence inter-
vals then were generated from the point estimates
for each of the variable loadings based on the
sampling distribution. The percentage variance
explained by principal component 1 was low
(39%) and confidence intervals for each loading
were large and encompassed zero (TABLE II)
suggesting that a single solution does not exist
for the combination of factors and their interac-
tion. Because a single solution does not appear to
exist stepwise procedures were performed for each
factor and their interaction independently.
357
Stepwise MANOVA was used to test treatment and
site effects and their interaction (Tabachnick and
Fidell 1996), with individual substrate activities as
dependant variables. Because maximum discrimina-
tion between main effects and their interaction was
desired we chose Rao’s V, which selects variables
based on their contribution to the overall separation
of groups (Tabachnick and Fidell 1996, Sobek and
Zak 2003). Five thousand bootstrap iterations were
performed for variable selection.
Stepwise Discriminant Function Analysis (DFA) was
performed on the grassland and oak-pine forest data,
again with individual substrate activities as dependent
variables and N treatment as the independent vari-
able. These DFA results are presented as a tool for
visualizing the separation of treatments in multivari-
ate space only as a visual complement to the
MANOVA results. For each DFA 5000 bootstrap
iterations were performed to select variables and to
build a sampling distribution against which the
observed data were compared. Confidence intervals
then were generated from the point estimates based
on the sampling distribution.
Criteria for evaluating and dealing with violations
to multivariate normality and homogeneous variance-
covariance matrices were adopted from Tabachnick
and Fidell (1996). Furthermore significance of
MANOVA was determined with Pillai’s trace, the
most robust, of the four test statistics used by SPSS to
calculate MANOVA P -values (Field 2000, Tabachnick
and Fidell 1996, Zar 1999). However test statistics
obtained with Wilk’s lambda and Hotelling’s trace
produced the same results. Post hoc analyses on
significant MANOVA either were obtained via all
possible combinations of two group MANOVA or via
univariate ANOVA. Bock (1975) suggested that these
Univariate ANOVA were protected by the original
MANOVA, but to be conservative type I error rate was
corrected with the sequential Bonferroni method
(Sokal and Rohlf 1995). When an ANOVA was found
to be significant it was followed up with Fisher’s LSD
post hoc tests. Type I error rate was controlled via
sequential Bonferroni method (Sokal and Rohlf
1995). All tests are considered significant at a 5
0.05 unless otherwise stated.
RESULTS
Glucose concentration and incubation time.—Results
of repeated-measures ANOVA indicated a signifi-
cant interaction between incubation time and
glucose concentrations on SA (P , 0.001) and SR
(P 5 0.001). LSD comparisons indicated that
5.0 g L21 of glucose at 120 h give the highest SA.
A significant difference between the levels of SA for
358
FIG. 1. Effects of glucose concentration and incubation
time on: A. Substrate Activity (sum of optical densities in all
wells per plate); B. Substrate Richness (number of utilized
nitrogen substrates). Values are means 6SE. Treatments
with the same letter designation are not significantly
different (a 5 0.05). x denotes 0.0 g glucose. e denotes
0.1 g glucose. % denotes 1.0 g glucose. D denotes 5.0 g
glucose. n 5 5.
the control (no glucose) and 0.1 g L21 addition was
present only at 72 h (FIG. 1a). According to LSD
tests maximum SR was reached with 1.0 g L21 and
5.0 g L21 of glucose at 120 h incubation (FIG. 1b).
Field test.—N plots. Two-way ANOVA of SA for the
Aug 2003 samples (first collection after initial
application of N) indicated that SA on N
compounds showed a significant interaction be-
tween sites and among N additions (P 5 0.047).
LSD comparisons indicated that the sotol grass-
land 4X treatment had the highest SA followed by
MYCOLOGIA
FIG. 2. Effects of locale and nitrogen fertilization on: A.
Substrate activity (sum of optical densities in all wells per
plate); B. Substrate richness (number of utilized nitrogen
substrates). Values are means 6SE. White 5 sotol grassland,
Gray 5 high elevation oak-pine forest. Overall substrate
activity was greatest in the sotol grassland. Treatments with
the same letter designation are not significantly different (a
5 0.05). n 5 10.
the sotol grassland 2X N application rate (FIG. 2a).
There were no significant differences in SA among
N application treatments for the oak-pine forest
site and the control plots in the grassland
(FIG. 2a). Furthermore SA is greatest overall in
the grassland. Substrate richness differed between
sites (P 5 0.009) and was highest in the grassland.
Substrate richness did not differ among N addi-
tions at either location (P 5 0.099) (FIG. 2b).
Nitrogen guilds SA and SR. Based on the two-way
MANOVA that examined N guild response SA for
the guilds of nitrogen substrates was significantly
 GRIZZLE AND ZAK: FUNGAL FUNCTIONAL DIVERSITY ON NITROGEN 359

different between sites (P , 0.001). The additions

of N had no significant affect on the SA for the
guilds of N compounds irrespective of site (P 5
0.762). Total SA for these guilds was highest in the
grassland. SA was significantly different between
treatments only for the amide guild (P 5 0.047),
substrate activity being higher in the 4X treatment
than in the X treatment regardless of site.
Based on the two-way ANOVA that examined N
guild response, SR for inorganic N (P , 0.001),
amides (P 5 0.040), nucleotides and nucleosides (P
5 0.047), L-amino acids (P 5 0.011), Di-amino acids
(P , 0.001) and miscellaneous (P , 0.001) guilds
were significantly different between sites. Substrate
richness for each of these guilds was highest in the
grassland.
Site discrimination. Results of stepwise MANOVA
showed a significant difference in fungal functional
diversity on nitrogen substrates between sites
(P , 0.001) with the grassland having the highest
N use (FIG. 5). Seven substrates were determined,
by stepwise procedure to account for differences
between the sites, c-amino-N-butyric acid, L-pyro-
glutamic acid, ethanolamine, agmatine, D-aspara-
gine, xanthosine and uridine. No guild was
dominant in the site analysis. Differences among
N additions were determined to be significant (P ,
0.001) based on stepwise MANOVA. These seven
substrates accounted for differences between N
treatments: L-histidine, L-cystine, L-methionine, L-
arginine, L-asparagine, uracil and thymine. L-
amino acids were the dominate guild in the
analysis. Across-site each of the N treatments were
significantly different based on two group MANO-
VAs with 4X having the highest SA followed by the
2X treatment. There was not a significant in-
teraction between site and treatment (P 5 0.204).
Only three substrates, L-pyroglutamic acid, c-
amino-N-butyric acid and ethanolamine, were used
in the MANOVA to evaluate the interaction FIG. 3. Graphical illustration of soil fungal functional
between site and amount of N addition because diversity in response to nitrogen additions in sotol grassland
addition of subsequent substrates in the analysis in Big Bend National Park, Texas, generated from
did not change the outcome of the analysis. Discriminant Function Analysis. A. Treatment separation
Within-site discrimination. Discriminant Function along DF 1 and DF 2 where the center (+) indicates the
Analysis for within-site differences due to N centroid of each treatment. Each centroid is encompassed
treatments on the grassland exhibited a high by 95% confidence ellipses. B. Discriminant function vector
degree of separation between treatments along loadings, where the numbered vector lines represent vector
discriminat function axes corresponding to N loadings of variables on DF1 and DF2 and correspond to
these nitrogen compounds on Biolog PM3 microtiter plates:
substrate use patterns (FIG. 3a). Discriminant
(1) uric acid, (2) glucuronamide, (3) L-homoserine, (4) N-
function (DF) 1 accounted for 88.4% of the acetyl glucosamine and (5) D-glutamic acid.
variation in the data, while DF 2 accounted for
11.6%. Five variables were included in the analysis:
uric acid, glucuronamide, L-homoserine, N-acetyl
galactosamine and D-glutamic acid. Each of the five
substrates represented a different guild, however
360 MYCOLOGIA

TABLE III. Correlations between nitrogen substrates and discriminant function axes obtained from Discriminant Function
Analysis. The data used in the analysis were the individual substrate activities of fungal assemblages from the Sotol Grasslands
and the high elevation Oak-Pine forest in Big Bend National Park. Numbers in parenthesis are percent variation explained. by
each discriminant function axes. Correlations are significant at P 5 0.05

Locationa Variableb Classc DF1(88.4%) DF2 (11.6%)

d
Sotol-Grasslands Uric acid Inorg N 0.782* 20.172
Glucuronamide Amides 0.085 0.294
L-homoserine L-amino acid 0.534* 0.387
N-acetyl glucosamine Amine 0.14 0.203
D-glutamic acid D-amino acid 20.233 20.579*
DF1(80.0%) DF2 (20.0%)
High Elevation N-acetyl galactosamine Amine 20.065 20.883*
Oak-Pine Forest L-cystine L-amino acid 0.625* 0.17
L-methionine L-amino acid 0.313 0.275
L-homoserine L-amino acid 20.272 0.482
Adenosine Nuce 20.218 0.422
a
Three treatments were evaluated in the Sotol-grasslands and at high-elevation Oak-Pine forest.
b
Nitrogen substrates from PM3 microtiter plates.
c
Guild of nitrogen substrates that have similar chemical makeup.
d
Inorganic nitrogen and simple organic nitrogen.
e
Nucleotides and nucleosides.
* Significant correlation.

two amino acids were included in the analysis (L-
homoserine and D-glutamic acid). Uric acid and L-
homoserine both were significantly correlated with
DF2 (TABLE III, FIG. 3b).
DFA for the oak-pine forest on N treatments
exhibited a high degree of separation between
treatments along discriminant function axes corre-
sponding to N substrate use patterns (FIG. 4a).
Discriminant function (DF) 1 accounted for 80.0%
of the variation in the data, while DF 2 accounted for
the remaining 20.0%. Five variables were included in
the analysis: L-cystine, N-acetyl galactosamine, L-
methionine, L-homoserine and adenosine. L-amino
acids were the dominant guild accounting for
differences in substrate use in the Oak-Pine forest.
L-cystine was significantly correlated with DF1 and N-
acetyl galactosamine with DF2. (TABLE III, FIG. 4b).
DISCUSSION
Glucose concentration and incubation time.—The
addition of glucose or some other carbon source
to the inoculum is necessary to activate the fungi
associated with the organic matter that is added to
the wells of the PM3 plates. In all preliminary trials
when no additional C was added to the inoculum
the fungi displayed growth only in those wells that
contain a suitable C source. For instance wells with
only NH4 or NO3 as the nitrogen source expressed
only minimal activity whereas the wells containing
amino acids expressed the greatest SA. Glucose
was chosen to activate N use because it requires no
induction period for fungal growth (Griffin 1994)
and has been used extensively as a means for
activating the microbial biomass when employing
the glucose stimulation technique for estimating
microbial biomass in soils. The amount of glucose
added to the wells should be only that amount
necessary to promote metabolic activity without
rapid luxuriant growth occurring. The goal during
method development and evaluation was to pro-
vide sufficient carbon to stimulate fungal activity
that would promote the uptake of the N source in
the well if fungi in the inoculum were capable. We
chose 1.0 g L21 glucose for our field trials because
maximum SR occurred with both 1.0 and
5.0 g L21 of glucose, but SA was lower with
1.0 g L21 than with 5.0 g L21. Therefore we hoped
to minimize the effect of glucose catabolism on
color development in the wells.
The need to add a C source to the PM3 microtiter
plates does provide the opportunity to evaluate
differences among sites or treatments not only in
response to N use but also with respect to the impact
of C type on fungal N use. For example, while we
added glucose as the activating C substrate, other
carbohydrates, carboxylic acids or recalcitrant sub-
strates could be added and differences in N use
evaluated in response to C availability.
Total SA and SR.—The field test at two sites showed
that the PM3 plates can distinguish differences in
fungal functional diversity between two vegetation
 GRIZZLE AND ZAK: FUNGAL FUNCTIONAL DIVERSITY ON NITROGEN
FIG. 4. Graphical illustration of soil fungal functional
diversity in response to nitrogen additions in the oak-pine
forest in Big Bend National Park, Texas, generated from
discriminant function analysis. A. Treatment separation
along DF 1 and DF 2 where the center (+) indicates the
centroid of each treatment. Each centroid is encompassed
by 95% confidence ellipses. B. Discriminant function vector
loadings, where the numbered vector lines represent vector
loadings of variables on DF1 and DF2 and correspond to
these nitrogen compounds on Biolog PM3 microtiter plates:
(1) L-cystine, (2) N-acetyl galactosamine, (3) L-methionine,
(4) L-homoserine and (5) adenosine.
361
FIG. 5. Graphical illustration of soil fungal functional
diversity on nitrogen substrates in response to nitrogen
additions in the oak-pine forest in Big Bend National Park,
Texas, generated from Discriminant Function Analysis. Bars
represent 95% confidence intervals.
zones based the rate of substrate use (SA) and the
number of N compounds used by each fungal
assemblage (SR). Furthermore the procedure
allowed for detection of shifts in function of
fungal communities between differing experimen-
tal N inputs within each site. The initial addition
of NH4+ and NO32 has not altered the fungal
community’s ability to use more compounds.
However the rate at which compounds were
metabolized in the 2X and 4X treatments in the
grassland was enhanced by the initial nitrogen
application. This suggests that N could be a limit-
ing factor for fungal growth in the grassland site
and the additional N inputs have stimulated
fungal metabolism.
Nitrogen guilds SA and SR.—The Nitrolog approach
distinguished between sites based on N guild use
data. Soil fungi must rely on the vegetation at each
site to supply substrates for growth (Cromack and
Caldwell 1992, Kjoller and Struwe 1992). More-
over vegetation affects N availability and sub-
sequent C : N ratios, which controls decomposition
rates. Therefore the observed differences in SA
and SR are likely due to the differences in plant
species composition between the sites.
Site, treatment and within-site discrimination.—The
Nitrolog procedure provided sufficient resolution
to discriminate between sites and treatments for N
substrate use by soil fungi at BBNP. The grassland
and forest sites were determined to be significantly
different from each other with respect to N use. Of
362 MYCOLOGIA
those compounds contributing to maximum
variation between sites several are either common-
ly found in grasses (L-pyroglutamic acid) or
accumulate in drought-stressed plants. c-amino-
N-butyric acid is a compound that has been found
to accumulate in plants during drought stress,
especially in the nodules of those associated with
nitrogen fixing bacteria (Merck 1996). Agmatine
is an intermediate in the putrescine synthesis
pathway using the enzyme arginine decarboxylase.
This pathway would be active during cell elonga-
tion and in nondividing mature tissues in plants
subjected to drought stress (Rastogi et al 1993,
Perez-Amador and Carbonell 1995). Mean activity
on each of these compounds was highest in the
SG. Because grasses are a dominant vegetation in
the SG site and drought stress is a common
occurrence there as well, especially during the
summer, these results suggest that the Nitrolog
procedure can discriminate between functional
differences of soil fungi that reflect specific
physiological adaptation to plants, and the effects
of chemical compounds exuded by them, to the
soil C and N pool within sites.
The Nitrolog method has applications to systems
where nitrogen metabolism is an important determi-
nate of fungal functional profiles, such as where
nitrogen is a limiting resource or where nitrogen
deposition is a concern. Furthermore combining N-
use data with C-use data should let the researcher
gain a more complete understanding of the overall
condition of an ecosystem. Defining a ratio of C vs. N
use would let the researcher monitor shifts in
requirements for each nutrient as disturbance alters
community resource needs.
Using Biolog plates to evaluate functional diversity in
microbial communities has generated some debate
based on limitations of and concerns with the pro-
cedure (Preston-Mafham et al 2002). While the
technique cannot characterize the species composition
of microbial communities, the usefulness of the
approach is in its ability to evaluate functional aspects
of microbial communities at different sites, subjected to
different treatments, or to follow responses to pertur-
bations over time. Furthermore, while Preson-Mafham
et al (2002) commented on the potential problems
related to inoculum densities in the microtiter plates,
this procedure is designed to maintain consistent
fungal inoculum, (Dobranic and Zak 1999, Sobek and
Zak 2003). Functional diversity of any ecosystem is an
emergent property that is determined by species
richness and species densities. Therefore changes in
functional diversity and the results of the Nitrolog
assessment will be determined by changes in species
density and composition over time.
To further examine the relationship between carbon
and nitrogen use by soil fungi and the effects on
ecosystem dynamics, other carbon substrates in addi-
tion to glucose could be added to the PM3 plates. In
this manner one could evaluate how patterns of root
exudates for example could affect the ability of the soil
fungal assemblage to use root-derived C and organic N
over the growing season in a quantifiable manner.
ACKNOWLEDGMENTS
Research was financed through a U.S.G.S.–B.R.D. Global
Climate Change Small Watershed Project grant and
a National Parks Service grant to Dr John Zak. Assistance
of the superintendent and staff at Big Bend National Park is
greatly appreciated. The authors thank Jim Campbell and
Dr Michael San Francisco for their help in discussing the
nitrogen substrates guilds. Field collections and laboratory
analysis were accomplished with the help of Jim Campbell,
Joseph Faust, Brandon Morris, Jennifer Resinger and
Kenneth Seal.
LITERATURE CITED
The Merck index: an encyclopedia of chemicals, drugs, and
biologicals. 12th ed. 1996. New Jersey: Merck & Co. Inc.
Arnebrant K, Baath E, Soderstrom B. 1990. Changes in
microfungal community structure after fertilization of
Scots Pine forest soil with ammonium nitrate or urea.
Soil Biol Biochem 22:309–312.
Baath E. 1988. A critical examination of the soil washing
technique with special reference to the effect of the size
of soil particles. Can J Bot 66:1566–1569.
Bardgett RD, Denton CS, Cook R. 1999. Below-ground
herbivory promotes soil nutrient transfer and root
growth in grassland. Ecol Letters 2:357–360.
———, Hobbs PJ, Frostegard A. 1996. Changes in soil
fungal: bacterial biomass ratios following reductions in
the intensity of management of an upland grassland.
Biol Fertil Soil 22:261–264.
———, Leemans DK. 1995. The short-term effects of
cessation of fertilizer applications, liming, and grazing
on microbial biomass and activity and activity in
a reseeded upland grassland soil. Biol Fertil Soil
19:148–154.
Bisset J, Widden P. 1972. An automatic, multichamber soil-
washing apparatus for removing fungal spores from
soil. Can J Microbiol 18:1399–1404.
Bock RD. 1975. Multivariate statistical methods in behav-
ioral research. New York: McGraw-Hill.
Carreiro MM, Sinsabaugh RL, Repert DA, Parkhurst DF.
2000. Microbial enzyme shifts explain litter decay
responses to simulated nitrogen deposition. Ecology
81:2359–2365.
Chapin FSI, Walker BH, Hobbs RJ, Hooper DU, Lawton JH,
Sala OE, Tilman D. 1997. Biotic control over the
functioning of ecosystems. Science 277:500–504.
Cook RC, Rayner ADM. 1984. Ecology of saprophytic fungi.
London and New York: Longman.
 GRIZZLE AND ZAK: FUNGAL FUNCTIONAL DIVERSITY ON NITROGEN
Cromack JK, Caldwell BA. 1992. The role of fungi in litter
decomposition and nutrient cycling. The fungal
community. New York: Marcel Dekker Inc. p 653–668.
DeForest JL, Zak DR, Pregitzer KS, Burton AJ. 2004.
Atmospheric nitrate deposition and the microbial
degradation of cellobiose and vanillin in a northern
hardwood forest. Soil Biol Biochem 36:965–971.
Dobranic JK, Zak J. 1999. A microtiter plate procedure for
evaluating fungal functional diversity. Mycologia
91:756–765.
Donnison LM, Griffith GS, Bardgett RD. 2000. Determi-
nants of fungal growth and activity in botanically
diverse hay meadows: effects of litter type and fertilizer
additions. Soil Biol Biochem 32:289–94.
Field A. 2000. Discovering statistics using SPSS for windows.
London: SAGE Publications. 496 p.
Garland JL, Mills AL. 1991. Classification and characteriza-
tion of heterotrophic microbial communities on the
basis of patterns of community-level sole-carbon-source
utilization. Appl Environment Microbiol 57:2351–2359.
Griffin DH. 1994. Fungal physiology. New York: John Wiley
& Sons Inc. 458 p.
Guckert JB, Carr GJ, Johnson TD, Hamm BG, Davidson DH,
Kumagai Y. 1996. Community analysis by biolog: curve
integration for statistical analysis of activated sludge
microbial habitats. J Microb Meth 27:183–187.
Haack SK, Garchow H, Klug MJ. 1995. Analysis of factors
affecting the accuracy, reproducibility, and interpreta-
tion of microbial community carbon source utilization
patterns. Appl Environ Microbiol 61:1458–1468.
Harley JL, Waid JS. 1955. A method of studying mycelia on
living roots and other surfaces in the soil. Trans Brit
Mycol Soc 38:104–118.
Hawksworth DL, Colwell RR. 1992. The fungal dimension of
biodiversity: magnitude, significance, and conservation.
Biodivers Conserv 1:221–226.
Hermann R, Stottlemyer R, Zak JC, Edmonds RL, VanMie-
groet H. 2000. Biogeochemical effects of global climate
change on U.S. National Parks. J Am Water Resource
Assoc 36:337–346.
Jenny H. 1980. The soil resource: origin and behavior. New
York: Springer Verlag. 377 p.
Kandeler E, Kampichler C, Horack O. 1996. Biology of
fertile soils. Biol Fertil Soil 23:299–306.
Kjoller A, Struwe S. 1992. Functional groups of microfungi
in decomposition. In: Carroll GC, Wicklow DT, eds.
The fungal community: its role and organization in the
ecosystem 2. New York: Marcel Dekker. p 619–630.
Parkinson D, Coleman D. 1991. Microbial communities,
activity and biomass. Ag, Ecosys Environ 34:3–33.
Pennanen T, Fritze H, Vanhala P, Kiikkila O, Neuvonen S,
Baath E. 1998. Structure of a microbial community in
soil after prolonged addition of low levels of simulated
acid rain. Appl Environ Microbiol 64:2173–2180.
Perez-Amador M, Carbonell J. 1995. Arginine decarboxylase
and putrescine oxidase in ovaries of Pisum sativum l.
363
Changes during ovary senescence and early stages of
fruit development. Plant Physiol 107:865–872.
Perry ADM, Amaranthus MP, Borchers JG, Borchero SL,
Brainerd RE. 1989. Bootstrapping in ecosystems. Bio-
science 39:230–237.
Preston-Mafham J, Boddy L, Randerson PF. 2002. Analysis
of microbial community functional diversity using sole-
carbon-source utilization profiles—a critique. Microb
Ecol 42:1–14.
Rastogi R, Dulson J, Rothstein S. 1993. Cloning of tomato
(Lycospersicon esculentum mill.) arginine decarboxylase
gene and its expression during fruit ripening. Plant
Physiol 103:829–834.
Smalla K, Wachtendorf U, Heuer H, Liu W-T, Forney L.
1998. Analysis of Biolog GN substrate utilization
patterns by microbial communities. Appl Environ
Microbiol 64:1220–1225.
Sobek E, Zak J. 2003. A microtiter plate method for
evaluating soil fungal functional diversity. Mycologia
95:590–602.
Sokal RR, Rohlf FJ. 1995. Biometry. New York: W.H.
Freeman & Co. 887 p.
Staff SS. 1985. Soil survey of Big Bend National Park. Part of
Brewster County: USDA, Soil Conservation Service.
Tabachnick BG, Fidell LS. 1996. Using multivariate statis-
tics. New York: HarperCollins. 880 p.
Vitousek PM, Aber JD, Howarth R, Likens GE, Matson PA,
Schindler D, Schlesinger WH, Tilman GD. 1997.
Human alteration of the global nitrogen cycle: causes
and consequences. Issues in Ecology.
Vogt KA, Bloomfield J, Ammirati J, Ammirati S. 1992.
Sporocarp production by basidiomycetes, with empha-
sis on forest ecosystems. In: Carroll GC, Wicklow DT,
eds. The fungal community: its role and organization
in the ecosystem 2. New York: Marcel Dekker. p 518–
563.
Willig MR, Moorhead DL, Cox SB, Zak J. 1996. Functional
diversity of soil bacterial communities in the Tabonuco
forest: interaction of anthropogenic and natural
disturbance. Biotropica 28:471–483.
Wolters V, Silver WL, Bignell DE, Coleman DC, Lavelle P,
Putten WH, Ruiter PD, Rusek J, Wall DH, Wardle DA,
Brussaard L, Dangerfield JM, Brown VK, Giller K,
Hooper DU, Sala O, Tiedje J, Veen JA. 2000. Effects of
global changes on above- and belowground biodiversity
in terrestrial ecosystems: implications for ecosystem
functioning. Bioscience 50:1089–1098.
Zak J, Willig MR, Moorhead DL, Wildman HG. 1994.
Functional diversity of microbial communities: a quan-
titative approach. Soil Biol Biochem 26:1101–1108.
Zar JH. 1999. Biostatistical analysis. New Jersey: Prentice
Hall. 663 p.
Zhang Q, Zak J. 1995. Effects of gapsize on litter
decomposition and microbial activity in a subtropical
forest. Ecology 76:2196–2204.