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Soil Biology & Biochemistry 38 (2006) 3431–3435
www.elsevier.com/locate/soilbio
Discrepancies between ergosterol and the phospholipid fatty acid
18:2o6,9 as biomarkers for fungi in boreal forest soils
Mona N. Högberg
Department of Forest Ecology, Swedish University of Agricultural Sciences, SLU, SE-901 83 Umeå, Sweden
Received 22 March 2006; received in revised form 29 May 2006; accepted 2 June 2006
Available online 10 July 2006
Abstract
Ergosterol and the phospholipid fatty acid (PLFA) 18:2o6,9 are frequently used as fungal biomarkers in studies on soils, and in
accordance with the ideal for biomarkers of microorganisms they are thought to turn over rapidly after cell death and lysis. These
biomarkers should also show the same patterns and responses to perturbations of the studied system. Here, I report strong correlations,
in natural boreal forests of contrasting fertility, between free ergosterol and PLFA 18:2o6,9 (r ¼ 0.821, P ¼ 0.007, n ¼ 9). Surprisingly,
ergosterol, but not PLFA 18:2o6,9, appears non-responsive to both large-scale tree girdling, which interrupts tree belowground C
allocation to ectomycorrhizal fungi, and to long-term N-loading, which may have negative effects on both mycorrhizal and saprotrophic
fungi. These results, therefore, question the use of ergosterol to monitor effects of soil perturbations on fungi in the field, but do not put
into question the use of the biomarker in natural forest ecosystems.
r 2006 Elsevier Ltd. All rights reserved.
Keywords: Boreal forests; Ergosterol; Fungal biomarkers; Mycorrhizal fungi; N fertilization; PLFA; Tree girdling
1. Introduction
The sterol ergosterol, ergosta-5,7,22-trien-3b-ol, and a
phospholipid fatty acid (PLFA), 18:2o6,9, are examples of
membrane-bound molecules commonly used as fungal
biomarkers in studies on soil (Ruzicka et al., 2000; Bååth
and Anderson, 2003, and references therein). The membrane
area is assumed to be well correlated with the bio-volume of
microbial cells, and as all ideal biomarkers of microorganisms
in soil they are thought to turn over rapidly after cell death
and lysis (Tunlid and White, 1992). Ideally, different markers
for the same microbial group should also show the same
patterns and responses to perturbations of the studied system.
That PLFA 18:2o6,9 is a good fungal biomarker in soils was
shown by Frostegård and Bååth (1996) when they found
strong positive correlations (r ¼ 0.92, n ¼ 11) between the
fungal specific ergosterol and PLFA 18:2o6,9 in soils from
cultivated fields, gardens, grasslands, and soils from beech
and spruce forests. The two biomarkers were also strongly
correlated in laboratory experiments studying composts
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doi:10.1016/j.soilbio.2006.06.002
consisting of mixtures of shredded straw and pig slurry,
during which saprophytic fungal biomass increased over time
(Klamer and Bååth, 2004). Another example of agreement
between the two fungal bio-markers was found in laboratory
incubations that aimed at reducing the abundance of
mycorrhizal fungi in forest soils but not that of the
saprophytic fungi (Bååth et al., 2004). Here, I tested (i)
whether ergosterol and fungal PLFAs show the same patterns
in undisturbed model forest ecosystems and (ii) whether they
respond similarly to N-fertilization or tree girdling, a
treatment which terminates the carbon supply to ectomycorrhizal fungi. I report large discrepancies between ergosterol
and PLFA 18:2o6,9 in their response to ecosystem perturbation in boreal forests, whereas in undisturbed boreal forest,
the biomarkers were strongly correlated.
2. Materials and methods
2.1. Study sites
The natural boreal forest is a 130-y-old forest at Betsele
northern Sweden (641390 N, 181300 E, 235 m altitude) with
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large variations in productivity, soil chemistry, and plant
community composition (Giesler et al., 1998; Högberg,
2001; Högberg et al., 2003, 2006a). This forest was sampled
along three transects, each encompassing a dwarf shrub
(DS) forest type (C:N ratio 38.174.1, pH 4.070.2,
5.574.1 mg inorganic N g1 organic matter (OM)), a short
herb (SH) forest type (C:N ratio 22.971.9, pH 4.670.1,
5.972.2 mg inorganic N g1 OM), and a tall herb (TH)
forest type (C:N ratio 14.970.6, soil pH 5.370.1,
19.3710.2 inorganic N g1 OM). OM concentration
(g OM g1 dry soil) decreased from 83% in the DS forest
type to 54% and 44% in the SH and TH forest types,
respectively.
I also used a large-scale tree-girdling experiment
(Högberg et al., 2001; Högberg and Högberg, 2002) at
Åheden, northern Sweden (641140 N, 191460 E, 175 m
altitude). This pine forest is classified as a DS forest type
(C:N ratio 37.972.2, pH 3.770.0, 2.270.9 mg inorganic
N g1 OM) and was subjected to tree girdling early (EG) or
late (LG) in the summer of 2000; there were three replicate
plots for each of these treatments and the control (C)
treatment. By girdling (cutting off the bark around the tree
at breast–height), the direct flow of photosynthate C to
roots and soil was terminated.
Lastly, I used a long-term N-loading experiment (Tamm,
1999; Högberg et al., 2006b) in a pine forest of DS forest
type (C:N ratio 37.572.1, pH 4.170.1, 1.270.5 mg
inorganic N g1 OM) at Norrliden, northern Sweden
(641210 N, 191450 E, 267 m altitude). Ammonium nitrate
was applied annually to plots (30 30 m) at four rates,
N0–N3, with three replicate plots per treatment. N0 is the
untreated control, which receives only the background N
deposition of ca. 3 kg ha1 y1, while N1 received additionally ca. 34 kg N ha1 y1, 1971–2004, and N2 twice the
N dose of N1, while N3 received ca. 108 kg ha1 y1
1971–1990, and is thus a high N treatment recovering from
the previous high N load (Högberg et al., 2006b).
2.2. Soil sampling
Soil was sampled on 18, 25, and 26 August 2004 at
Betsele, Norrliden, and Åheden, respectively. In each case,
i.e., at a location along transects at Betsele or a plot in the
experiments, three samples of the organic mor-layer (F+H
horizons, approximately corresponding to Oe+Oa) were
taken with a 0.15-m (diameter) corer and bulked together
to represent one replicate. I quickly and carefully sorted
out roots by passing the soil through a sieve (5 mm mesh
size). A maximum time period of 3 h at 11–14 1C preceded
storage on dry ice (78 1C). Thereafter, samples were kept
in a freezer (20 1C). Sub-samples for standard chemical
characterization of the soils were analysed fresh, as
described below. At Betsele, sampling was done along the
three separate transects (N ¼ 3), each encompassing all
three forest types. Thus, composited soil samples were
collected in the DS forest type at three positions (at 0, 10,
and 20 m distance, n ¼ 3), similarly in the SH forest type
(at 40, 50, 60 m distance) and in the TH forest type (at 80,
85, and 90 m distance). At Norrliden and Åheden, three
plots of each treatment were sampled; the central 400 and
100 m2 area of each plot was sampled randomly at
Norrliden and Åheden, respectively. The mean values of
three transects through each forest type (site Betsele) and
the mean values of three plots of each treatment (sites
Norrliden and Åheden) were used in the statistical analysis
(N ¼ 3).
2.3. Analysis
Soil pH was measured at a soil:water ratio of 1:3 (v v1).
Dried soil samples (70 1C, 84 h) were analysed for C% and
N% using a CN analyser coupled to a mass spectrometer
(Ohlsson and Wallmark, 1999). Organic matter content of
the mor-layer, extractable ammonium and nitrate were
analysed as given in Högberg et al. (2006a). Free ergosterol
was extracted from 10 mg root-free, freeze-dried and milled
soil material. The sample was vortexed intensively together
with 0.5 ml ethanol for 2 min, and then centrifuged at
11 400 g for 30 min. The extraction step was repeated twice
and 50 ml of the resulting supernatant was injected and
analysed in an HPLC system (Sundberg et al., 1999). The
analysis was performed at the laboratory of Prof. Torgny
Näsholm, Department of Forest Genetics and Plant
Physiology, SLU, S-901 83 Umeå, Sweden. Analysis of
fungal-specific PLFAs in soil (Tunlid and White, 1992;
Frostegård and Bååth, 1996) followed the Bligh and Dyer
(1959) method as modified by Frostegård et al. (1991,
1993). Lipids were extracted from frozen soil equivalent to
0.15 g OM and separated on silica gel columns (Bond Elut
LRC, SI 100 mg, Varian, Palo Alto, CA, USA). Lipids
were eluted in sequence with chloroform, acetone, and
methanol. The chloroform fraction (containing neutral
lipids, NLFAs) and the methanol fraction (containing
PLFAs) were dried under N2, dissolved and subjected to
mild methanolysis. The resulting fatty acid methyl esters
were analysed on a GC (PDZ Europa Ltd., Northwich,
Chesire, England), equipped with a 25 m phenyl methyl
siloxane column (internal diameter, 0.2 mm; film thickness,
0.33 mm) (Ultra 2 column, Agilent Technologies, Palo Alto,
CA, USA). The identity of individual PLFA peaks were
determined by comparing retention times of authentic
FAME standards (FAME 37-47885-U, Supelco, Bellefonte, USA), and by comparing the retention times for
individual PLFA peaks with peaks that had been identified
by GC–MS. Methylnonadecanoic acid (Me19:0) was used
as internal standard. Fatty acids are designated as the total
number of C atoms:number of double bonds, followed by
the positions of the double bonds. Two PLFAs were used
as indicators of fungal biomass: 18:2o6,9 (Federle, 1986;
Frostegård and Bååth, 1996; Olsson, 1999) and 18:1o9
(Bååth, 2003). The full data set on 18:2o6,9 and 18:1o9
and other PLFA biomarkers is reported in a study on the
general microbial community structure at the same sites
and experiments as studied here (Högberg M.N. et al.,
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substantial reductions under conditions of high N load.
For example, PLFA 18:2o6,9 was reduced by 62% in N1
plots as compared to N0 plots and by 72% in N2 as
compared to N0 plots. Similar declines were observed for
350
350
Ergosterol
PLFA 18:1ω9
PLFA 18:2ω6,9
(a)
300
3. Results
Fig. 1. Abundance of the fungal biomarkers ergosterol and PLFAs
18:2o6,9 and 18:1o9 in boreal forest soils (a) in undisturbed forests with
increasing forest productivity from a DS, through an SH to a TH forest
type; (b) in an experimental forest in which the trees were girdled
(C, control plots; EG, early girdled plots, girdled in June 2000; LG, late
girdled plots, girdled in August 2000); and (c) in an experimental forest
with 34 years of N-load (N0, control plots; N1 and N2, annual loads of 34
and 68 kg N ha1 y1, respectively; N3, plots recovering since 14 years
from a previous load of 108 kg N ha1 y1 during 20 years). Mean71SEM, N ¼ 3.
250
250
200
200
150
150
100
100
50
50
0
0
DS
SH
TH
350
350
(b)
Ergosterol (µg g-1 organic matter)
The soil ergosterol concentration in the three undisturbed and low-productive DS forests, i.e., the DS forest at
Betsele, C plots at Åheden, and N0 plots at Norrliden, did
not differ significantly (P ¼ 0.662); the concentration
varied between 243 and 346 mg g1 OM with a mean of
274715 mg g1 OM. Similarly, there were small differences
in the concentration of PLFA 18:2o6,9 between the DS
forest types at the three sites; concentrations varied
between 185 and 258 nmol g1 OM. The mean was
210742 nmol g1 OM. Nor did PLFA 18:1o9, another
eukaryotic biomarker, reveal any differences between the
DS forest types at the different sites. PLFAs 18:2o6,9 and
18:1o9 were strongly correlated among the three natural
DS forests in the study (r ¼ 0.823, P ¼ 0.006).
The concentration of ergosterol at Betsele decreased by
44% from the DS to the intermediate SH and by 72% from
the SH to the TH forest type, while that of PLFA 18:2o6,9
also decreased by 44% from the DS to the SH forest type,
and by 69% from the SH to the TH forest type (P ¼ 0.001)
(Fig. 1a), which represents a remarkable agreement. Thus,
ergosterol was strongly correlated to PLFA 18:2o6,9
(r ¼ 0.821, P ¼ 0.007). Both ergosterol content (r ¼
0.446, P ¼ 0.229) and PLFA 18:2o6,9 content (r ¼ 0.659,
P ¼ 0.053) were uncorrelated to the other PLFA fungal
biomarker, 18:1o9, although percentages of total amount
of PLFAs (molar percentages) of PLFAs 18:2o6,9 and
18:1o9 were strongly correlated.
However, in a soil to which the C supply to ectomycorrhizal fungi had been terminated 4 years ago by girdling of
the trees, ergosterol showed no effect of the treatment,
while PLFA 18:2o6,9 showed a reduction by 45%, and
PLFA 18:1o9 showed a smaller negative response to the
treatment (Fig. 1b). The two fungal PLFAs were correlated
(r ¼ 0.872, P ¼ 0.002). Also, in the case of the N-loading
experiment, ergosterol showed no response at all to the Nloading treatments (Fig. 1c), while both PLFAs showed
300
300
300
250
250
200
200
150
150
100
100
50
50
0
0
C
EG
LG
350
350
(c)
300
300
250
250
200
200
150
150
100
100
50
50
0
0
N0
N1
N2
N3
Forest type or treatment
PLFA (nmol g-1 organic matter)
in press). I used one-way analysis of variance followed by
Tukey’s multiple comparison tests to test for differences
between the DS forest types at Betsele, Norrliden, and
Åheden. Pearson’s product moment correlation was used
for tests of correlations (n ¼ 9, unless at Norrliden where
n ¼ 12). Means are reported 71 standard deviation.
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PLFA 18:1o9 with declines of 48% and 60% in N1 and N2
plots, respectively, as compared to N0 plots. The two
PLFAs were thus strongly correlated across N treatments
at Norrliden (r ¼ 0.977, Po0.000).
4. Discussion
Ergosterol values (Fig. 1) in the low-productive DS
forests are considerably higher than those frequently found
in low-C soils (Grant and West, 1986; West et al., 1987;
Djajakirana et al., 1996; Frostegård and Bååth, 1996;
Ruzicka et al., 2000), but are similar to those commonly
found in Fennoscandian coniferous forests rich in OM
(Fritze et al., 1994; Smolander et al., 1994; Pietikäinen and
Fritze, 1995; Frostegård and Bååth, 1996). The two
eukaryotic PLFAs 18:2o6,9 and 18:1o9 were strongly
correlated in the DS forests, which supports their use as
fungal biomarkers in these forests. In contrast, all three
biomarkers showed considerable variability across the
undisturbed soil pH and N supply (and forest productivity)
gradients at Betsele (Fig. 1a). Similar dramatic differences
in PLFA 18:2o6,9 content among the forest types at
Betsele were also observed in June 1994 (Högberg et al.,
2003) and in October 2000 (Nilsson et al., 2005), and the
pattern observed here therefore seems stable.
However, in the relatively long-term (4–34 years)
perturbation experiments studied here, ergosterol showed
no response to the treatments, while PLFAs indicative of
fungi, especially 18:2o6,9, responded strongly. Notably,
both fungal PLFAs showed tendencies to be higher in the
terminated N3 treatment than in the on-going N2
treatment, which is in agreement with other signs of
ecosystem recovery in the N3 treatment (Quist et al., 1999;
Högberg et al., 2006b).
It was recently shown that the decomposition of
ergosterol, in soil or plant litter without living fungi, but
with (or without) bacterial assemblages, was a rather slow
process with a half-life of ca. 3–5 months (Mille-Lindblom
et al., 2004), and added ergosterol was little if at all
decomposed during a 10-day incubation (Zhao et al., 2005,
2006). It is generally held that fungi are the main actors in
the process of decomposition of OM (e.g., Killham, 1994,
p. 51). At Norrliden, we know that the soil OM
decomposition is negatively affected by N additions
(Franklin et al., 2003), which, at least partly, could be
explained by the lower abundance of fungi according to
PLFA analysis. Unfortunately at Åheden, we have as yet
no information of changes in the decompostion of OM.
However, we know that soil respiration rates in the girdled
plots are still 50% lower than in the control plots (reflecting
the absence of respiration by ectomycorrhizal roots and
fungi).
My results cast doubts over the use of ergosterol in field
studies of forest ecosystem perturbations on soil fungi.
Ergosterol appears suitable under relatively stable soil
conditions, as shown here along the undisturbed Betsele
forest productivity gradient and in studies by Frostegård
and Bååth (1996), Ruzicka et al. (2000), and others. In the
very first studies on ergosterol as a fungal biomarker in
soil, its use was strongly recommended to quantify changes
in the fungal populations of soils (Grant and West, 1986;
West et al., 1987). Ergosterol was later successfully used to
study growth (increases in fungal biomass) of ectomycorrhizal or saprophytic fungi (Ekblad et al., 1995; Bååth,
2001; Zhao et al., 2005, 2006), or decreases in fungi during
storage of soils in the laboratory (West et al., 1987; Bååth
et al., 2004). In a recent laboratory study, the decomposition of ergosterol did not mirror the decline in fungal
biomass C after additions of bactericides, fungicides,
herbicides, or chloroform fumigation (Zhao et al., 2005,
2006). As was shown here, ergosterol apparently fails to
detect fundamental negative treatment effects in the field,
clearly revealed by fungal PLFA biomarkers, and in the
case of the tree-girdling experiment, also evident as an
almost total elimination of sporocarp production by
ectomycorrhizal fungi (Högberg et al., 2001), and as a
substantial reduction in microbial biomass C (Högberg and
Högberg, 2002).
5. Conclusions
I suggest that ergosterol is a good biomarker for fungi in
relatively undisturbed microbial communities in boreal
forests, but does not appear to indicate detrimental effects
of forest ecosystem perturbations, clearly revealed by
analysis of fungal PLFAs.
Acknowledgements
I thank Professor T. Näsholm and M. Zetherström
(Department of Forest Genetics and Plant Physiology,
SLU, Umeå, Sweden) for performing the HPLC analyses. I
thank Dr Rockie Yarwood (Department of Corp and Soil
Science, OSU, Corvallis, USA) for advice on the PLFA
analysis. This study was financially supported by the
Swedish Research Council for Environment, Agricultural
Sciences and Spatial Planning (FORMAS), and the
Swedish Science Council (VR).
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