Temporal changes in diversity and expres
Soil Biology & Biochemistry 41 (2009) 1380–1389
Contents lists available at ScienceDirect
Soil Biology & Biochemistry
journal homepage: www.elsevier.com/locate/soilbio
Temporal changes in diversity and expression patterns of fungal laccase genes
within the organic horizon of a brown forest soil
Harald Kellner a, b, *, Patricia Luis c, Bettina Schlitt a, b, François Buscot a, b
a
UFZ - Helmholtz Centre for Environmental Research (Leipzig-Halle) Ltd., Department of Soil Ecology, Theodor-Lieser-Str. 4, D-06120 Halle/Saale, Germany
University of Leipzig, Institute of Botany - Terrestrial Ecology, Johannisallee 21, D-04103 Leipzig, Germany
c
ˆtiment Andre
´ Lwoff, 43 Boulevard du 11 Novembre 1918,
Universite´ de Lyon, Universite´ Lyon 1, Ecologie Microbienne, UMR CNRS 5557, USC INRA 1193, Ba
F-69622 Villeurbanne Cedex, France
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 December 2008
Received in revised form
10 March 2009
Accepted 23 March 2009
Available online 21 April 2009
Temporal fluctuations of present and expressed fungal genes encoding the ligninolytic enzyme laccase
were examined bimonthly between March 2004 and April 2005 in the organic horizon of a beech forest
Cambisol. Using two sets of degenerate primer pairs, we detected 73 different basidiomycete laccase
genes from soil DNA extracts and 42 different transcripts of asco- and basidiomycetes from soil RNA
extracts (cDNA). Phylogenetic analysis related the sequences to fungal taxa. The highest fungal laccase
gene richness in soil DNA and RNA samples were found in August, October and January, and followed the
input of fresh litter into soil. The highest change of the fungal laccase gene population was observed from
October to January, but no distinct temporal change in the total soil laccase activity was measured.
Present and expressed laccase gene populations were highly different, implying different subsets
amplified with our primer sets and likely impacting future research strategies. Despite considerable
variations in gene presence and expression, we found a steady expression and high soil enzyme activity
of fungal laccases at each sampling date, thus presumably laccases have major impacts to soil organic
matter turnover and stabilization processes.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Ascomycetes
Basidiomycetes
Laccase gene expression
Lignin degradation
1. Introduction
The accumulation and turnover of soil organic matter (SOM)
result from a balance between mineralization and stabilization due
to the recalcitrance of some compounds and the formation of low
bioavailable organo-mineral complexes (von Lu¨tzow et al., 2006).
Soil microorganisms are largely involved in both mineralization
and stabilization processes, and in forest ecosystems fungi are key
players (Berg and McClaugherty, 2003; Osono, 2007). In deciduous
forests, plant litter is one major annual resource input for soil fungal
communities, and it is colonized in relatively short time scales
(Berg and Gerstberger, 2004). The first colonizers, i.e. yeasts,
zygomycetes and several ascomycetes, use easy available soluble
compounds before being replaced by asco- and basidiomycetes able
to attack insoluble and more recalcitrant substances such as
cellulose and lignin (Dighton, 1997; Koide et al., 2005). Recently,
* Corresponding author. FUSAGx, Unite´ de Biologie animale et microbienne,
Avenue Mare´chal Juin 6, B-5030 Gembloux, Belgium. Tel.: þ32 81 622355; fax: þ32
81 611555.
E-mail address: mail@haraldkellner.com (H. Kellner).
URL: http://www.haraldkellner.com/html/laccase_project.html
0038-0717/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.soilbio.2009.03.012
molecular techniques allow a better understanding and monitoring
of these fungal dynamics directly in soil. In a recent study on the
degradation of leaf litter in a native and foreign habitat, it was
demonstrated that the general degradation capability was site
specific and probably driven by fungal adaptation to the local leaf
litter type (Aneja et al., 2006). Thus, degradation of sugars, starch,
cellulose and lignin by the native communities was higher. In
course of the degradation, the typical increase in the proportion of
lignin was observed (Aneja et al., 2006).
Lignin, the second most abundant biopolymer in nature, is
largely resistant against microbial attack (Ko¨gel-Knabner, 2002) but
is degraded by fungi producing ligninolytic oxidative exoenzymes
(Thorn, 1997). Monitoring changes in fungal communities that
produce such exoenzymes provides information on their potential
role in temporal SOM degradation processes. Instead of monitoring
fungal species based on their ribosomal RNA genes or spacer
regions, studies following this line should analyze genes encoding
exoenzymes such as phenol oxidases (laccases), manganese- and
lignin-peroxidases, b-glucosidases or cellobiohydrolases that
degrade plant litter (Zak et al., 2006; Edwards et al., 2008).
Within fungal oxidative exoenzymes, laccases are one of the
most universal. They catalyze the oxidation of various aromatic
H. Kellner et al. / Soil Biology & Biochemistry 41 (2009) 1380–1389
1381
compounds, particularly phenolic substrates, coupled to the
reduction of oxygen to water and are highly involved in biodegradation of biopolymers such as lignin (Baldrian, 2006). It was shown
that the diversity of basidiomycete laccase genes and the total soil
laccase activity declined parallel with the SOM content along
a vertical soil profile of a beech/oak-forest Cambisol (Luis et al.,
2004, 2005b). In the present study, an analysis of the temporal
change of basidiomycetes harboring laccase genes was conducted
in the same forest soil at six sampling dates during one year. This
study considered both the gene presence and their expression, and
also measured the laccase activity in the soil. A previous work had
shown a high spatial heterogeneity in diversity and expression of
the genes (Luis et al., 2005a,b), hence this study was performed at
a more homogeneous place of the stand in a pure beech trees
plantation (Fagus sylvatica).
We focused our study on the organic forest floor layer that
harbors the highest laccase gene diversity and laccase activity at the
site (Luis et al., 2005a,b) and which is submitted to the highest
temporal variations concerning litter input, soil temperature,
metric potential which all might influence the composition and
activity of ligninolytic soil fungi. We hypothesized that temporal
variations in gene presence, gene expression and activity of
resulting enzymes would not necessarily be synchronous and
would have distinct orders of magnitude.
were located within a homogenous stand of beech trees (the
nearest oak tree was at 12 m) without underground vegetation and
had a minimum distance to a tree or to the next plot of 3 m.
According to previous results, this distance was found to correspond
to independent samples (Luis et al., 2005b). One plot had an area of
20 20 cm, which should be highly dependent in case of threedimensional growing fungi. Within this plot area (20 20 cm),
three soil cores (subsamples) with 2 cm diameter and approximately 10 cm deep, were taken at each date, the organic layer
including the litter was separated, all subsamples were mixed to
a composite sample and stored immediately in liquid nitrogen. The
opened holes were locked with a 15 ml centrifuge tube to remain
the soil as undisturbed as possible. This sampling process was
repeated on each sampling date for each plot. During the sampling
campaign, fruiting bodies (if found) were collected for subsequent
molecular analysis. The fruiting bodies were dried and deposited at
the herbarium of the University of Leipzig, Germany (LZ).
2. Materials and methods
2.3. PCR, sequencing and sequence analysis
2.1. Sampling site and design
Basidiomycete laccase genes from soil DNA and soil RNA
(cDNA) samples were amplified using the degenerate primer pair
Cu1F (50 -CAY TGG CAY GGN TTY TTY CA-30 ) and Cu2R (50 -G RCT
GTG GTA CCA GAA NGT NCC-30 ) (Luis et al., 2004). Furthermore,
the degenerate primer pair Cu1AF (50 -ACM WCB GTY CAY TGG CAY
GG-30 ) and Cu2R was used to amplify ascomycete laccase genes
from the soil RNA (cDNA) samples (Kellner et al., 2007a). In order
to determine general fungal growing activity, the fungal b-tubulin
gene was amplified from soil RNA (cDNA) using the primer pair
B3.6F and B1.2R (Thon and Royse, 1999). All amplification settings
followed protocols of Luis et al. (2004, 2005a) and Kellner et al.
(2007a).
From each amplification product (3 soil samples per date), 7 ml
was loaded onto a 2% agarose gel and electrophoresed twice, for
technical replication. Thereafter, the gels were stained with
ethidium bromide and obtained DNA bands were visualized and
photographed under UV light. Subsequently, the intensity of the
DNA bands derived from the expression analysis of laccase and btubulin genes were quantified using ImageQuant version 5.0 Software (Molecular Dynamics, Amersham Biosciences). All amplified
laccase gene products were corrected against the intensity of the
appropriate b-tubulin gene intensity. The highest corrected quantification value for the laccase PCR products was set to 100.
All laccase gene PCR products derived from soil DNA and soil
RNA (cDNA) were then cloned and sequenced using the protocols of
Luis et al. (2005b). Nucleotide sequences were compared with the
Soil samples were taken from an integrated experimental station
¨ K; University of Bayrof the Institute of Ecosystem Research (BITO
euth) located in the Steigerwald (49 520 2600 N, 10 270 5400 E) near
Bamberg (Bavaria, Germany) at an elevation of 460 m above the sea
level. The site is covered by a 100-year-old mixed stand of European
beech (F. sylvatica L.) and Pedunculate oak (Quercus robur L.) with
sparse understorey vegetation. The soil is a Dystric Cambisol (FAOUNESCO, 1990) characterized by a fine moder humus layer and a C:N
ratio of 20. The pH is 4.2 for the organic layer. The turnover of organic
matter in this soil type is rapid with a low accumulation in the lower
horizons. The organic O-horizon contains about 40% of organic
carbon (Kaiser et al., 2002). The climate is cool-temperate with
a transition from oceanic to continental, an average temperature of
7.0–8.0 C and 850 mm annual precipitation. Climatic parameters
¨ K; University of Bayreuth) and
were continuously measured (BITO
mean values of the one-week time-span before the sampling are
given in Table 1. Soil water content was measured by following the
mass loss of 10 g organic horizon soil composite sample, dried for
24 h at 90 C. The peak period of litterfall is between October and
November (Kalbitz, personal communication).
Samples of the organic layer, including fresh litter, were
collected in three small plots (named P2, P4, P6 on the site) during
one year: March, June, August, October 2004 and January, April
2005 (March 2004–April 2005, Table 1). The three sampled plots
2.2. Soil DNA and RNA extraction, cDNA synthesis
The DNA extraction from the soil samples was performed
according to Luis et al. (2004). Total RNA was extracted from the
same homogenized soil samples and cDNAs were synthesized
following the procedures described in Luis et al. (2005a).
Table 1
Climatic parameters and total soil water content for each sampling date.
Sampling date
Air temperature ( C)a
Soil temperature ( C)b
Rainfall before sampling (mm)a
Soil water content (%)
March 2004
June 2004
August 2004
October 2004
January 2005
April 2005
8.8
12.5
15.3
5.2
4.5
8.2
7.0
11.0
15.6
8.8
5.0
7.8
17.5
14.0
25.0
4.0
4.0
3.0
72.9
59.3
63.2
63.3
72.8
67.1
a
b
5.0
3.8
3.9
2.1
2.1
4.2
1.58
0.9
1.01
0.5
0.52
1.42
Mean s.d. for week before sampling; data every 10 min (n ¼ 1008).
Mean s.d. for week before sampling; data every 1 h (n ¼ 168); O-horizon.
1382
H. Kellner et al. / Soil Biology & Biochemistry 41 (2009) 1380–1389
databases of the National Center for Biotechnology Information
(NCBI) using the Blast search algorithm (Altschul et al., 1997). The
sequences, which gave the best blast matches, including putative
multicopper oxidases from the Clusters of Orthologous Groups of
proteins KOG1263 (Tatusov et al., 2003), were retrieved and integrated into the phylogenetic analysis.
The sequences were aligned using BioEdit7 (Hall, 1999) and the
final nucleotide alignment, available online under study no. S2321 in
TreeBASE
(http://www.treebase.org/treebase/index.html),
was
exported to a NEXUS file and analyzed using PAUP*4.0b10 (Swofford,
1998). A neighbor-joining (NJ) tree was constructed using the
Kimura 2-parameter model. All laccase gene sequences of this
analysis were submitted to GenBank and are available under
accession numbers EF423278–EF423319 and EF439882–EF439898.
the differences among the population, the integrated UniFrac
cluster analysis was used and the integrated UniFrac P Test was
used for calculation of statistical significance (Lozupone, 2005).
2.5. Laccase activity in soil samples
Twenty soil samples of the organic layer surrounding the three
plots were taken each sampling date and analyzed for laccase
activity using ABTS [2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonate); Sigma] as described in Luis et al. (2005b). The activity was
expressed in units (1 U ¼ 1 mmol min1) per gram of dry soil matter.
Statistical significance between sequential samplings was conducted using SigmaStat 2.03 (SPSS Inc., US).
3. Results
2.4. Data and population analysis
3.1. Sequence analysis of fungal laccase genes
The different types of laccase genes, their eventual affiliation to
taxa and functional groups (i.e. saprotrophic or mycorrhizal fungi)
were determined from the constructed neighbor-joining (NJ) tree
and recorded for each plot (i.e. P2, P4, P6) and sampling month in
a quantitative matrix. Sequence differences were recorded on the
level of the nucleic acids; intron appearance, and high branch
bootstrap values gave additional support for the taxonomic relationship of the soil DNA derived sequences. All detected laccase
genes, having the same DNA sequence, were set as identical laccase
gene ‘‘type’’. The different types were analyzed for dominance
among all analyzed plots, and their richness (S), diversity (H) and
evenness (E), including clone numbers, were also estimated for
each plot. The diversity index (H), including the different gene
types and their clone number, was estimated using the Shannon
equation [H ¼ S(Pi*ln(Pi))] (Shannon, 1948). The evenness (E),
which is a combination of the Shannon index and the richness
coefficient, was calculated using the formula [E ¼ H/ln(S)]. The
richness and diversity were tested for significant differences among
the sampling dates by one-way ANOVA (SigmaStat 2.03, SPSS Inc.,
US). All conclusions were based on at least the 5% level of significance (P < 0.05).
To estimate the temporal variation of the fungal laccase gene
populations, two approaches were used: (1) a presence/absence
analysis of the laccase genes characterized by Sørensen distance
amongst two sequential sampling dates using the program PC-Ord
4.41 (McCune and Mefford, 1999; Luis et al., 2005b). The Sørensen
distance, measured as percent dissimilarity, is a proportion coefficient given by the formula [1 2W/(A þ B)]; where W is the sum of
shared abundances and A & B are the sums of abundances in
individual sample units (Beals, 1984). And (2) a genetic population
analysis of all laccase gene sequences per sampling date (combined
data of the three plots) using the program Arlequin 3.01 (Excoffier
et al., 2005) and UniFrac (Lozupone, 2005, see also Lauber et al.,
2009). For analysis of the genetic population using Arlequin, each
recorded different laccase gene sequence was treated as haplotype.
To compare the genetic variation between the populations,
dissimilarity indices (FST) (Reynolds et al., 1983; Slatkin, 1995)
between all pairs of populations for laccase types amplified from
soil DNA and the combined soil RNA (cDNA) were estimated. The
statistical significance of these genetic distances was tested by
10,000 permutations of the haplotypes between the populations.
Moreover, a laccase gene alignment without references but
including an outgroup sequence was used for constructing
a maximum likelihood phylogenetic tree, and uploaded into the
UniFrac population difference software (Lozupone, 2005). UniFrac
is a metric of phylogenetic divergence that calculates the genetic
distance between multiple gene libraries as the percentage of
branch length that leads to only one of a pair of samples. To show
Fragments of fungal laccase genes were amplified from soil DNA
extracts using degenerate primer pairs annealing in the conserved
copper binding regions I and II (cbr I and II, Fig. 1). The amplicons
ranged from 140 bp to 200 bp. A total of 450 clones obtained from
PCR products of soil DNA extracts were analyzed (3 plots 6
sampling dates 25 clones). The primer pair Cu1F/Cu2R yielded in
408 laccase genes fragments from the O-horizon, which were
separated in 73 distinct genes (Fig. 2). The primer pair Cu1AF/Cu2R
also gave amplicons, but the obtained sequences were not related
to fungal laccase genes.
The analysis of 180 expressed gene sequences obtained with
both primer pairs from soil RNA extracts (cDNAs) revealed 42
different laccase gene types. Noteworthy, only five expressed genes
corresponded to those amplified from the DNA extracts.
A neighbor-joining analysis of all detected laccase genes clustered
the sequences in major clades representing different fungal families
or orders like Agaricales sensu lato, Atheliaceae, Boletales, Mycenaceae, Russulaceae (Fig. 2). Most sequences could be related to
basidiomycetes while only few ascomycete laccase genes were
found (Fig. 2). Major clades containing Lactarius spp., Mycena spp.,
Piloderma spp. and Russula spp. received bootstrap support (Fig. 2).
The intron structure of 165 laccase genes obtained from soil DNA
extracts and collected fruit body-DNA was analyzed between copper
binding region I and II (Fig. 1). Eighty-eight genes (46 soil derived, 42
of fungal references) comprised one intron except Macrotyphula
juncea (AJ542615) and Mycena rosea (AJ542628) that had two
between cbr I and cbr II (Table 2). Further 77 laccase gene types, 27
from soil and 50 from reference fungi, had no intron. Thirteen
different intron positions were detected (Fig. 1, Table 2) and all
introns followed the GT/AG rule. Some intron insertion sites were
specific as they were found for members of distinct clades (Fig. 2).
Most Russulaceae comprised one intron at gene fragment position
121. Mycena species clustering in three subclades had clade specific
introns at positions 45, 62 and 109, respectively (Fig. 2, Table 2).
3.2. Fungal laccase gene dominance structure in soil samples
Out of 73 different laccase gene types amplified from soil DNA
extracts, 34 were found at least two times in different soil samples.
Moreover, 14 gene types were found more than four times and
considered as dominant in the study (Fig. 3). Among these dominant types, 6 belonged to saprotrophic fungi of the species Mycena
cinerella (the most dominant in this investigation), Mycena spp.,
Hemimycena spp. and Bovista nigrescens, whereas 4 were related to
mycorrhizal species of the Russulaceae (Figs. 2 and 3). The last 4
dominant types could not be related to any species or genera and
were treated as unknown type with unknown trophic status. The
H. Kellner et al. / Soil Biology & Biochemistry 41 (2009) 1380–1389
1383
Fig. 1. Schematic representation of a typical fungal laccase between copper binding regions cbr I and cbr II. Numbers beneath show the different found introns and their insertion
position. Boxes marked with ‘‘ins’’ and ‘‘del’’ indicate positions, where amino acid insertion or deletions was found (see also supplementary information in Hassett et al., 2009).
dominant fungal laccase genes related to B. nigrescens, Hemimycena
sp., Lactarius sp., M. cinerella, Russula fellea and Russula mairei/
ochroleuca were found throughout the whole year (Fig. 3).
A total of 11 sequences from the soil DNA extracts were identical
to amplicons from fruiting bodies collected at the sampling site
(Fig. 3), highlighting the high identification rate of soil-derived
sequences in this study. Of the 73 different laccase gene types found
in the soil, 21 (29%) were related to saprotrophic and 22 (30%) to
mycorrhizal fungi; which were based on phylogeny, bootstrap
support of distinct clades, and intron appearance (Fig. 2). The
remaining 30 (41%) types could be related neither to taxa nor to
a trophic status (‘‘unknown’’).
A dominance study on the expressed laccase genes showed 10
different genes to be expressed at least in two soil samples, while
Fig. 2. Neighbor-joining tree calculated from the coding region of fungal laccase genes obtained from soil samples as well as fungal references using K2P-distances. Branch support
was assessed by 2000 bootstrap replicates, which are given above branches. Occurrence of introns supporting fungal taxa is indicated. Major monophyletic fungal groups are
indicated with bold branches, their trophic status is indicated (m, mycorrhizal; s, saprotrophic; u, unknown). The occurrence of each environmental laccase gene sequence is given
in brackets (D1–D6 indicating sampling dates March 2004–April 2005; sampling plots: P2, P4, P6) with their accordingly GenBank accession number. Expressed laccase genes, high
ranked ‘‘1’’ refers to a sequence obtained with primer combination Cu1Af/Cu2r, whereas ‘‘2’’ indicates the amplification with both primer sets and without number means the
finding only with primer set Cu1f/Cu2r.
1384
H. Kellner et al. / Soil Biology & Biochemistry 41 (2009) 1380–1389
Table 2
Characterization of the intron occurrence in all analyzed laccase sequences, giving position, length, reading frame and fungi including it.
Intron no. No. of laccase gene Insertion
(see Fig. 1) types including it
position
Length of
Frame Fungal references including it
intron (bp)
1
2
3
4
5
6
7
8
9
10
11
12
13
No intron
71
49–55
51–53
51
51–55
53–60
52
61
49
53
52–58
53–61
52–63
–
1
11
2
1
6
6
2
1
1
1
5
10
44
77
26
45
51
53
62
64
65
80
81
92
109
118
121
þ1
þ2
þ2
þ1
þ1
0
þ1
þ1
þ2
þ1
0
0
0
–
Macrotyphula juncea
Mycena cinerella, Mycena crocata, Mycena galopus, Mycena zephirus
Pleurotus ostreatus
Botryotinia fuckeliana
Mycena zephirus
Rhizoctonia solani
Macrotyphula juncea, Mycena rosea
Unknown
Unknown
Mycena rosea
Mycena crocata, Mycena galopus
Bovista nigrescens, Hemimycena crispata, Piloderma byssinum, Pseudocyphellaria crocata, Tylospora fibrillosa
Hemimycena crispata, all Russula sp., all Lactarius sp.
Nearly all Ascomycota, many Basidomycota (except Mycena and Russulaceae species)
the remaining 32 gene types were only found as singletons. Out of
these 42 gene types, 28 (67%) were related to basidio- and 14 (33%)
to ascomycete taxa (Fig. 2). The most dominant expressed gene
type, with an appearance of four times in either different plots or on
a different sampling date, was related to B. nigrescens.
3.3. Temporal variation of basidiomycete laccase gene diversity
In a first step, the temporal variations in abundance of
basidiomycete laccase genes were analyzed (for each unique type
to a maximum appearance of 3, if found in all 3 plots). An
increase from March (29 laccase genes) to October 2004 (45
genes), which corresponded to an enhanced detection of saprotrophic and unknown basidiomycetes, was followed by a decrease
in January and April 2005 (33 and 37 genes) (Fig. 4a, Table 3). The
Shannon diversity indices, including the clone number of individual sequence types found at each sampling date varied
consistently with the richness of the laccase gene population
(Table 3). Thus, the highest Shannon diversity of the community
was in October 2004 with 3.182, whereas the lowest was
Fig. 3. Dominance structure of environmental basidiomycete laccase gene types found with a minimum appearance of three times among all samples analyzed. Genes found as
singletons and doubletons were only shown if they had identical fungal references or were found to be expressed. Accordingly GenBank accession numbers, sampling date,
sampling frequency (appearance in one to three plots), relation to laccase genes of fungal reference strains and their possible trophic status (myc – mycorrhizal, sap – saprotrophic)
are given. Species marked with * were collected with fruiting bodies at the forest site. Laccase gene types marked with ‘‘E’’ were also found expressed.
H. Kellner et al. / Soil Biology & Biochemistry 41 (2009) 1380–1389
observed in March, June 2004 and January 2005 (2.528–2.877)
(Table 3). The evenness of the fungal laccase gene population
reached values between 0.785 and 0.917, indicating an even
distribution of the sequenced clones among the different gene
types (Table 3). However, the mean richness and mean diversity
indices of the 3 soil replicates (plots) per sampling date (Table 3)
were not significant different among all sampling dates. The
highest dissimilarity between two consecutive samplings was
measured between October and January, representing a change of
more than 50% of the laccase genes present on these two
sampling dates (Fig. 4b). Dissimilarities among other sampling
dates ranged between 15 and 30% (Fig. 4b).
In a further step, the genetic difference between the laccase
gene populations was investigated. No significant difference
(UniFrac P Test and Arlequin FST; P < 0.05 significance level) of
the laccase gene populations obtained from genomic DNA
between different sampling dates was found (Table 4). The
clustering of the laccase gene populations amplified from soil
DNA showed no consistent trend between consecutive samplings,
except a clustering of the January and April 2005 populations
apart from the four other laccase gene populations obtained from
soil DNA extracts (Fig. 5).
1385
3.4. Expression of fungal laccase genes in soil samples
From RNA extracts that were processed in cDNAs, 18 and 33
different laccase gene types were amplified with the primer pair
combinations Cu1F/Cu2R and Cu1AF/Cu2R, respectively. As 9 gene
types were amplified with both primer pairs, altogether 42
different laccase genes were detected in the expression study. Gene
expression was observed at all sampling dates (Fig. 6a). The highest
relative expression rates, using both primer sets, were found in
August 2004 and January 2005 (Fig. 6a). The highest richness of
expressed genes was observed in January 2005 with 11 different
types per primer set, whereby some genes were revealed with both
primer sets. In August 2004, 11 different laccase genes were also
obtained but only with primer pair Cu1AF/Cu2R. A clearer picture of
the expression pattern could be drawn when analyzing the total
richness per sampling date, i.e. the total amount of expressed gene
types detected with both primer sets (‘‘S’’ in Fig. 6a). With 19
different expressed gene types, the highest richness was observed
in January 2005, followed by August and October 2004 (11), March
2004 (9), June 2004 (4) and April 2005 (2) (Fig. 6a). The attempt to
attribute the expressed genes to fungal groups and taxa via cluster
analysis (Fig. 2) showed expression shifts between asco- and
basidiomycetes. In January 2005, 16 of the expressed genes
belonged to basidio- and 3 to ascomycetes, whereas in October
2004, 7 different genes were expressed in asco- vs. 4 in basidiomycetes (Fig. 6a). The strong relative expression found in August
2004 corresponded to 7 different basidio- and 4 different ascomycete laccase genes (Fig. 6a).
Out of 28 expressed basidiomycete laccase genes, 13 (46%) could
be attributed to precise taxonomic levels, i.e. Agaricales sensu lato,
Atheliaceae, Sebacinaceae, B. nigrescens (Fig. 2). In January 2005, 7
Table 3
Number of sequences obtained from each single plot and plots combined for each
sampling date (community), analyzed for laccase gene richness (S), evenness (E) and
Shannon diversity index (H). Numbering of single soil cores followed sampling date
(March 2004–April 2005) and plot name (P2, P4, P6). Numbers in brackets for pooled
laccase gene richness include the abundances of each type found among three
possible plots.
Sampling analysis
Single plots
March 2004 P2
March 2004 P4
March 2004 P6
June 2004 P2
June 2004 P4
June 2004 P6
August 2004 P2
August 2004 P4
August 2004 P6
October 2004 P2
October 2004 P4
October 2004 P6
January 2005 P2
January 2005 P4
January 2005 P6
April 2005 P2
April 2005 P4
April 2005 P6
Fig. 4. Characterization of the basidiomycete laccase gene abundance separated by
putative nutritional status (a), and their change (dissimilarity) between two consecutive samplings according their presence and absence (b).
No. of sequences
22
21
25
17
23
20
24
26
25
23
27
26
21
24
19
23
25
17
Pooled data of 3 plots – ‘‘pooled community’’
March 2004
68
June 2004
60
August 2004
75
October 2004
76
January 2005
64
April 2005
65
Whole campaign
408
Laccase gene population
S
E
H
8
9
12
10
11
10
14
10
14
16
20
9
7
15
11
16
11
10
0.806
0.929
0.889
0.934
0.921
0.928
0.961
0.875
0.923
0.931
0.967
0.887
0.785
0.9
0.889
0.971
0.931
0.919
1.677
2.042
2.209
2.15
2.209
2.138
2.535
2.014
2.435
2.582
2.898
1.949
1.527
2.437
2.132
2.691
2.232
2.115
21 (29)
23 (31)
28 (38)
34 (45)
25 (33)
26 (37)
73
0.879
0.917
0.909
0.902
0.785
0.906
0.831
2.677
2.877
3.028
3.182
2.528
2.951
3.564
1386
H. Kellner et al. / Soil Biology & Biochemistry 41 (2009) 1380–1389
Table 4
Statistic significance (P values) of difference among the temporal soil DNA amplified laccase gene populations (DNA March 2004–April 2005) and the expressed laccase genes
(RNA all). Above diagonal, P values of UniFrac P Test and below diagonal, population differentiation based on 10,000 permutations (pairwise differences) using Arlequin. Bold
values indicate the significant different populations (P < 0.05).
DNA March 2004
DNA March 2004
DNA June 2004
DNA August 2004
DNA October 2004
DNA January 2005
DNA April 2005
RNA all
DNA June 2004
0.96
0.82
0.86
0.81
0.42
0.44
Contents lists available at ScienceDirect
Soil Biology & Biochemistry
journal homepage: www.elsevier.com/locate/soilbio
Temporal changes in diversity and expression patterns of fungal laccase genes
within the organic horizon of a brown forest soil
Harald Kellner a, b, *, Patricia Luis c, Bettina Schlitt a, b, François Buscot a, b
a
UFZ - Helmholtz Centre for Environmental Research (Leipzig-Halle) Ltd., Department of Soil Ecology, Theodor-Lieser-Str. 4, D-06120 Halle/Saale, Germany
University of Leipzig, Institute of Botany - Terrestrial Ecology, Johannisallee 21, D-04103 Leipzig, Germany
c
ˆtiment Andre
´ Lwoff, 43 Boulevard du 11 Novembre 1918,
Universite´ de Lyon, Universite´ Lyon 1, Ecologie Microbienne, UMR CNRS 5557, USC INRA 1193, Ba
F-69622 Villeurbanne Cedex, France
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 December 2008
Received in revised form
10 March 2009
Accepted 23 March 2009
Available online 21 April 2009
Temporal fluctuations of present and expressed fungal genes encoding the ligninolytic enzyme laccase
were examined bimonthly between March 2004 and April 2005 in the organic horizon of a beech forest
Cambisol. Using two sets of degenerate primer pairs, we detected 73 different basidiomycete laccase
genes from soil DNA extracts and 42 different transcripts of asco- and basidiomycetes from soil RNA
extracts (cDNA). Phylogenetic analysis related the sequences to fungal taxa. The highest fungal laccase
gene richness in soil DNA and RNA samples were found in August, October and January, and followed the
input of fresh litter into soil. The highest change of the fungal laccase gene population was observed from
October to January, but no distinct temporal change in the total soil laccase activity was measured.
Present and expressed laccase gene populations were highly different, implying different subsets
amplified with our primer sets and likely impacting future research strategies. Despite considerable
variations in gene presence and expression, we found a steady expression and high soil enzyme activity
of fungal laccases at each sampling date, thus presumably laccases have major impacts to soil organic
matter turnover and stabilization processes.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Ascomycetes
Basidiomycetes
Laccase gene expression
Lignin degradation
1. Introduction
The accumulation and turnover of soil organic matter (SOM)
result from a balance between mineralization and stabilization due
to the recalcitrance of some compounds and the formation of low
bioavailable organo-mineral complexes (von Lu¨tzow et al., 2006).
Soil microorganisms are largely involved in both mineralization
and stabilization processes, and in forest ecosystems fungi are key
players (Berg and McClaugherty, 2003; Osono, 2007). In deciduous
forests, plant litter is one major annual resource input for soil fungal
communities, and it is colonized in relatively short time scales
(Berg and Gerstberger, 2004). The first colonizers, i.e. yeasts,
zygomycetes and several ascomycetes, use easy available soluble
compounds before being replaced by asco- and basidiomycetes able
to attack insoluble and more recalcitrant substances such as
cellulose and lignin (Dighton, 1997; Koide et al., 2005). Recently,
* Corresponding author. FUSAGx, Unite´ de Biologie animale et microbienne,
Avenue Mare´chal Juin 6, B-5030 Gembloux, Belgium. Tel.: þ32 81 622355; fax: þ32
81 611555.
E-mail address: mail@haraldkellner.com (H. Kellner).
URL: http://www.haraldkellner.com/html/laccase_project.html
0038-0717/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.soilbio.2009.03.012
molecular techniques allow a better understanding and monitoring
of these fungal dynamics directly in soil. In a recent study on the
degradation of leaf litter in a native and foreign habitat, it was
demonstrated that the general degradation capability was site
specific and probably driven by fungal adaptation to the local leaf
litter type (Aneja et al., 2006). Thus, degradation of sugars, starch,
cellulose and lignin by the native communities was higher. In
course of the degradation, the typical increase in the proportion of
lignin was observed (Aneja et al., 2006).
Lignin, the second most abundant biopolymer in nature, is
largely resistant against microbial attack (Ko¨gel-Knabner, 2002) but
is degraded by fungi producing ligninolytic oxidative exoenzymes
(Thorn, 1997). Monitoring changes in fungal communities that
produce such exoenzymes provides information on their potential
role in temporal SOM degradation processes. Instead of monitoring
fungal species based on their ribosomal RNA genes or spacer
regions, studies following this line should analyze genes encoding
exoenzymes such as phenol oxidases (laccases), manganese- and
lignin-peroxidases, b-glucosidases or cellobiohydrolases that
degrade plant litter (Zak et al., 2006; Edwards et al., 2008).
Within fungal oxidative exoenzymes, laccases are one of the
most universal. They catalyze the oxidation of various aromatic
H. Kellner et al. / Soil Biology & Biochemistry 41 (2009) 1380–1389
1381
compounds, particularly phenolic substrates, coupled to the
reduction of oxygen to water and are highly involved in biodegradation of biopolymers such as lignin (Baldrian, 2006). It was shown
that the diversity of basidiomycete laccase genes and the total soil
laccase activity declined parallel with the SOM content along
a vertical soil profile of a beech/oak-forest Cambisol (Luis et al.,
2004, 2005b). In the present study, an analysis of the temporal
change of basidiomycetes harboring laccase genes was conducted
in the same forest soil at six sampling dates during one year. This
study considered both the gene presence and their expression, and
also measured the laccase activity in the soil. A previous work had
shown a high spatial heterogeneity in diversity and expression of
the genes (Luis et al., 2005a,b), hence this study was performed at
a more homogeneous place of the stand in a pure beech trees
plantation (Fagus sylvatica).
We focused our study on the organic forest floor layer that
harbors the highest laccase gene diversity and laccase activity at the
site (Luis et al., 2005a,b) and which is submitted to the highest
temporal variations concerning litter input, soil temperature,
metric potential which all might influence the composition and
activity of ligninolytic soil fungi. We hypothesized that temporal
variations in gene presence, gene expression and activity of
resulting enzymes would not necessarily be synchronous and
would have distinct orders of magnitude.
were located within a homogenous stand of beech trees (the
nearest oak tree was at 12 m) without underground vegetation and
had a minimum distance to a tree or to the next plot of 3 m.
According to previous results, this distance was found to correspond
to independent samples (Luis et al., 2005b). One plot had an area of
20 20 cm, which should be highly dependent in case of threedimensional growing fungi. Within this plot area (20 20 cm),
three soil cores (subsamples) with 2 cm diameter and approximately 10 cm deep, were taken at each date, the organic layer
including the litter was separated, all subsamples were mixed to
a composite sample and stored immediately in liquid nitrogen. The
opened holes were locked with a 15 ml centrifuge tube to remain
the soil as undisturbed as possible. This sampling process was
repeated on each sampling date for each plot. During the sampling
campaign, fruiting bodies (if found) were collected for subsequent
molecular analysis. The fruiting bodies were dried and deposited at
the herbarium of the University of Leipzig, Germany (LZ).
2. Materials and methods
2.3. PCR, sequencing and sequence analysis
2.1. Sampling site and design
Basidiomycete laccase genes from soil DNA and soil RNA
(cDNA) samples were amplified using the degenerate primer pair
Cu1F (50 -CAY TGG CAY GGN TTY TTY CA-30 ) and Cu2R (50 -G RCT
GTG GTA CCA GAA NGT NCC-30 ) (Luis et al., 2004). Furthermore,
the degenerate primer pair Cu1AF (50 -ACM WCB GTY CAY TGG CAY
GG-30 ) and Cu2R was used to amplify ascomycete laccase genes
from the soil RNA (cDNA) samples (Kellner et al., 2007a). In order
to determine general fungal growing activity, the fungal b-tubulin
gene was amplified from soil RNA (cDNA) using the primer pair
B3.6F and B1.2R (Thon and Royse, 1999). All amplification settings
followed protocols of Luis et al. (2004, 2005a) and Kellner et al.
(2007a).
From each amplification product (3 soil samples per date), 7 ml
was loaded onto a 2% agarose gel and electrophoresed twice, for
technical replication. Thereafter, the gels were stained with
ethidium bromide and obtained DNA bands were visualized and
photographed under UV light. Subsequently, the intensity of the
DNA bands derived from the expression analysis of laccase and btubulin genes were quantified using ImageQuant version 5.0 Software (Molecular Dynamics, Amersham Biosciences). All amplified
laccase gene products were corrected against the intensity of the
appropriate b-tubulin gene intensity. The highest corrected quantification value for the laccase PCR products was set to 100.
All laccase gene PCR products derived from soil DNA and soil
RNA (cDNA) were then cloned and sequenced using the protocols of
Luis et al. (2005b). Nucleotide sequences were compared with the
Soil samples were taken from an integrated experimental station
¨ K; University of Bayrof the Institute of Ecosystem Research (BITO
euth) located in the Steigerwald (49 520 2600 N, 10 270 5400 E) near
Bamberg (Bavaria, Germany) at an elevation of 460 m above the sea
level. The site is covered by a 100-year-old mixed stand of European
beech (F. sylvatica L.) and Pedunculate oak (Quercus robur L.) with
sparse understorey vegetation. The soil is a Dystric Cambisol (FAOUNESCO, 1990) characterized by a fine moder humus layer and a C:N
ratio of 20. The pH is 4.2 for the organic layer. The turnover of organic
matter in this soil type is rapid with a low accumulation in the lower
horizons. The organic O-horizon contains about 40% of organic
carbon (Kaiser et al., 2002). The climate is cool-temperate with
a transition from oceanic to continental, an average temperature of
7.0–8.0 C and 850 mm annual precipitation. Climatic parameters
¨ K; University of Bayreuth) and
were continuously measured (BITO
mean values of the one-week time-span before the sampling are
given in Table 1. Soil water content was measured by following the
mass loss of 10 g organic horizon soil composite sample, dried for
24 h at 90 C. The peak period of litterfall is between October and
November (Kalbitz, personal communication).
Samples of the organic layer, including fresh litter, were
collected in three small plots (named P2, P4, P6 on the site) during
one year: March, June, August, October 2004 and January, April
2005 (March 2004–April 2005, Table 1). The three sampled plots
2.2. Soil DNA and RNA extraction, cDNA synthesis
The DNA extraction from the soil samples was performed
according to Luis et al. (2004). Total RNA was extracted from the
same homogenized soil samples and cDNAs were synthesized
following the procedures described in Luis et al. (2005a).
Table 1
Climatic parameters and total soil water content for each sampling date.
Sampling date
Air temperature ( C)a
Soil temperature ( C)b
Rainfall before sampling (mm)a
Soil water content (%)
March 2004
June 2004
August 2004
October 2004
January 2005
April 2005
8.8
12.5
15.3
5.2
4.5
8.2
7.0
11.0
15.6
8.8
5.0
7.8
17.5
14.0
25.0
4.0
4.0
3.0
72.9
59.3
63.2
63.3
72.8
67.1
a
b
5.0
3.8
3.9
2.1
2.1
4.2
1.58
0.9
1.01
0.5
0.52
1.42
Mean s.d. for week before sampling; data every 10 min (n ¼ 1008).
Mean s.d. for week before sampling; data every 1 h (n ¼ 168); O-horizon.
1382
H. Kellner et al. / Soil Biology & Biochemistry 41 (2009) 1380–1389
databases of the National Center for Biotechnology Information
(NCBI) using the Blast search algorithm (Altschul et al., 1997). The
sequences, which gave the best blast matches, including putative
multicopper oxidases from the Clusters of Orthologous Groups of
proteins KOG1263 (Tatusov et al., 2003), were retrieved and integrated into the phylogenetic analysis.
The sequences were aligned using BioEdit7 (Hall, 1999) and the
final nucleotide alignment, available online under study no. S2321 in
TreeBASE
(http://www.treebase.org/treebase/index.html),
was
exported to a NEXUS file and analyzed using PAUP*4.0b10 (Swofford,
1998). A neighbor-joining (NJ) tree was constructed using the
Kimura 2-parameter model. All laccase gene sequences of this
analysis were submitted to GenBank and are available under
accession numbers EF423278–EF423319 and EF439882–EF439898.
the differences among the population, the integrated UniFrac
cluster analysis was used and the integrated UniFrac P Test was
used for calculation of statistical significance (Lozupone, 2005).
2.5. Laccase activity in soil samples
Twenty soil samples of the organic layer surrounding the three
plots were taken each sampling date and analyzed for laccase
activity using ABTS [2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonate); Sigma] as described in Luis et al. (2005b). The activity was
expressed in units (1 U ¼ 1 mmol min1) per gram of dry soil matter.
Statistical significance between sequential samplings was conducted using SigmaStat 2.03 (SPSS Inc., US).
3. Results
2.4. Data and population analysis
3.1. Sequence analysis of fungal laccase genes
The different types of laccase genes, their eventual affiliation to
taxa and functional groups (i.e. saprotrophic or mycorrhizal fungi)
were determined from the constructed neighbor-joining (NJ) tree
and recorded for each plot (i.e. P2, P4, P6) and sampling month in
a quantitative matrix. Sequence differences were recorded on the
level of the nucleic acids; intron appearance, and high branch
bootstrap values gave additional support for the taxonomic relationship of the soil DNA derived sequences. All detected laccase
genes, having the same DNA sequence, were set as identical laccase
gene ‘‘type’’. The different types were analyzed for dominance
among all analyzed plots, and their richness (S), diversity (H) and
evenness (E), including clone numbers, were also estimated for
each plot. The diversity index (H), including the different gene
types and their clone number, was estimated using the Shannon
equation [H ¼ S(Pi*ln(Pi))] (Shannon, 1948). The evenness (E),
which is a combination of the Shannon index and the richness
coefficient, was calculated using the formula [E ¼ H/ln(S)]. The
richness and diversity were tested for significant differences among
the sampling dates by one-way ANOVA (SigmaStat 2.03, SPSS Inc.,
US). All conclusions were based on at least the 5% level of significance (P < 0.05).
To estimate the temporal variation of the fungal laccase gene
populations, two approaches were used: (1) a presence/absence
analysis of the laccase genes characterized by Sørensen distance
amongst two sequential sampling dates using the program PC-Ord
4.41 (McCune and Mefford, 1999; Luis et al., 2005b). The Sørensen
distance, measured as percent dissimilarity, is a proportion coefficient given by the formula [1 2W/(A þ B)]; where W is the sum of
shared abundances and A & B are the sums of abundances in
individual sample units (Beals, 1984). And (2) a genetic population
analysis of all laccase gene sequences per sampling date (combined
data of the three plots) using the program Arlequin 3.01 (Excoffier
et al., 2005) and UniFrac (Lozupone, 2005, see also Lauber et al.,
2009). For analysis of the genetic population using Arlequin, each
recorded different laccase gene sequence was treated as haplotype.
To compare the genetic variation between the populations,
dissimilarity indices (FST) (Reynolds et al., 1983; Slatkin, 1995)
between all pairs of populations for laccase types amplified from
soil DNA and the combined soil RNA (cDNA) were estimated. The
statistical significance of these genetic distances was tested by
10,000 permutations of the haplotypes between the populations.
Moreover, a laccase gene alignment without references but
including an outgroup sequence was used for constructing
a maximum likelihood phylogenetic tree, and uploaded into the
UniFrac population difference software (Lozupone, 2005). UniFrac
is a metric of phylogenetic divergence that calculates the genetic
distance between multiple gene libraries as the percentage of
branch length that leads to only one of a pair of samples. To show
Fragments of fungal laccase genes were amplified from soil DNA
extracts using degenerate primer pairs annealing in the conserved
copper binding regions I and II (cbr I and II, Fig. 1). The amplicons
ranged from 140 bp to 200 bp. A total of 450 clones obtained from
PCR products of soil DNA extracts were analyzed (3 plots 6
sampling dates 25 clones). The primer pair Cu1F/Cu2R yielded in
408 laccase genes fragments from the O-horizon, which were
separated in 73 distinct genes (Fig. 2). The primer pair Cu1AF/Cu2R
also gave amplicons, but the obtained sequences were not related
to fungal laccase genes.
The analysis of 180 expressed gene sequences obtained with
both primer pairs from soil RNA extracts (cDNAs) revealed 42
different laccase gene types. Noteworthy, only five expressed genes
corresponded to those amplified from the DNA extracts.
A neighbor-joining analysis of all detected laccase genes clustered
the sequences in major clades representing different fungal families
or orders like Agaricales sensu lato, Atheliaceae, Boletales, Mycenaceae, Russulaceae (Fig. 2). Most sequences could be related to
basidiomycetes while only few ascomycete laccase genes were
found (Fig. 2). Major clades containing Lactarius spp., Mycena spp.,
Piloderma spp. and Russula spp. received bootstrap support (Fig. 2).
The intron structure of 165 laccase genes obtained from soil DNA
extracts and collected fruit body-DNA was analyzed between copper
binding region I and II (Fig. 1). Eighty-eight genes (46 soil derived, 42
of fungal references) comprised one intron except Macrotyphula
juncea (AJ542615) and Mycena rosea (AJ542628) that had two
between cbr I and cbr II (Table 2). Further 77 laccase gene types, 27
from soil and 50 from reference fungi, had no intron. Thirteen
different intron positions were detected (Fig. 1, Table 2) and all
introns followed the GT/AG rule. Some intron insertion sites were
specific as they were found for members of distinct clades (Fig. 2).
Most Russulaceae comprised one intron at gene fragment position
121. Mycena species clustering in three subclades had clade specific
introns at positions 45, 62 and 109, respectively (Fig. 2, Table 2).
3.2. Fungal laccase gene dominance structure in soil samples
Out of 73 different laccase gene types amplified from soil DNA
extracts, 34 were found at least two times in different soil samples.
Moreover, 14 gene types were found more than four times and
considered as dominant in the study (Fig. 3). Among these dominant types, 6 belonged to saprotrophic fungi of the species Mycena
cinerella (the most dominant in this investigation), Mycena spp.,
Hemimycena spp. and Bovista nigrescens, whereas 4 were related to
mycorrhizal species of the Russulaceae (Figs. 2 and 3). The last 4
dominant types could not be related to any species or genera and
were treated as unknown type with unknown trophic status. The
H. Kellner et al. / Soil Biology & Biochemistry 41 (2009) 1380–1389
1383
Fig. 1. Schematic representation of a typical fungal laccase between copper binding regions cbr I and cbr II. Numbers beneath show the different found introns and their insertion
position. Boxes marked with ‘‘ins’’ and ‘‘del’’ indicate positions, where amino acid insertion or deletions was found (see also supplementary information in Hassett et al., 2009).
dominant fungal laccase genes related to B. nigrescens, Hemimycena
sp., Lactarius sp., M. cinerella, Russula fellea and Russula mairei/
ochroleuca were found throughout the whole year (Fig. 3).
A total of 11 sequences from the soil DNA extracts were identical
to amplicons from fruiting bodies collected at the sampling site
(Fig. 3), highlighting the high identification rate of soil-derived
sequences in this study. Of the 73 different laccase gene types found
in the soil, 21 (29%) were related to saprotrophic and 22 (30%) to
mycorrhizal fungi; which were based on phylogeny, bootstrap
support of distinct clades, and intron appearance (Fig. 2). The
remaining 30 (41%) types could be related neither to taxa nor to
a trophic status (‘‘unknown’’).
A dominance study on the expressed laccase genes showed 10
different genes to be expressed at least in two soil samples, while
Fig. 2. Neighbor-joining tree calculated from the coding region of fungal laccase genes obtained from soil samples as well as fungal references using K2P-distances. Branch support
was assessed by 2000 bootstrap replicates, which are given above branches. Occurrence of introns supporting fungal taxa is indicated. Major monophyletic fungal groups are
indicated with bold branches, their trophic status is indicated (m, mycorrhizal; s, saprotrophic; u, unknown). The occurrence of each environmental laccase gene sequence is given
in brackets (D1–D6 indicating sampling dates March 2004–April 2005; sampling plots: P2, P4, P6) with their accordingly GenBank accession number. Expressed laccase genes, high
ranked ‘‘1’’ refers to a sequence obtained with primer combination Cu1Af/Cu2r, whereas ‘‘2’’ indicates the amplification with both primer sets and without number means the
finding only with primer set Cu1f/Cu2r.
1384
H. Kellner et al. / Soil Biology & Biochemistry 41 (2009) 1380–1389
Table 2
Characterization of the intron occurrence in all analyzed laccase sequences, giving position, length, reading frame and fungi including it.
Intron no. No. of laccase gene Insertion
(see Fig. 1) types including it
position
Length of
Frame Fungal references including it
intron (bp)
1
2
3
4
5
6
7
8
9
10
11
12
13
No intron
71
49–55
51–53
51
51–55
53–60
52
61
49
53
52–58
53–61
52–63
–
1
11
2
1
6
6
2
1
1
1
5
10
44
77
26
45
51
53
62
64
65
80
81
92
109
118
121
þ1
þ2
þ2
þ1
þ1
0
þ1
þ1
þ2
þ1
0
0
0
–
Macrotyphula juncea
Mycena cinerella, Mycena crocata, Mycena galopus, Mycena zephirus
Pleurotus ostreatus
Botryotinia fuckeliana
Mycena zephirus
Rhizoctonia solani
Macrotyphula juncea, Mycena rosea
Unknown
Unknown
Mycena rosea
Mycena crocata, Mycena galopus
Bovista nigrescens, Hemimycena crispata, Piloderma byssinum, Pseudocyphellaria crocata, Tylospora fibrillosa
Hemimycena crispata, all Russula sp., all Lactarius sp.
Nearly all Ascomycota, many Basidomycota (except Mycena and Russulaceae species)
the remaining 32 gene types were only found as singletons. Out of
these 42 gene types, 28 (67%) were related to basidio- and 14 (33%)
to ascomycete taxa (Fig. 2). The most dominant expressed gene
type, with an appearance of four times in either different plots or on
a different sampling date, was related to B. nigrescens.
3.3. Temporal variation of basidiomycete laccase gene diversity
In a first step, the temporal variations in abundance of
basidiomycete laccase genes were analyzed (for each unique type
to a maximum appearance of 3, if found in all 3 plots). An
increase from March (29 laccase genes) to October 2004 (45
genes), which corresponded to an enhanced detection of saprotrophic and unknown basidiomycetes, was followed by a decrease
in January and April 2005 (33 and 37 genes) (Fig. 4a, Table 3). The
Shannon diversity indices, including the clone number of individual sequence types found at each sampling date varied
consistently with the richness of the laccase gene population
(Table 3). Thus, the highest Shannon diversity of the community
was in October 2004 with 3.182, whereas the lowest was
Fig. 3. Dominance structure of environmental basidiomycete laccase gene types found with a minimum appearance of three times among all samples analyzed. Genes found as
singletons and doubletons were only shown if they had identical fungal references or were found to be expressed. Accordingly GenBank accession numbers, sampling date,
sampling frequency (appearance in one to three plots), relation to laccase genes of fungal reference strains and their possible trophic status (myc – mycorrhizal, sap – saprotrophic)
are given. Species marked with * were collected with fruiting bodies at the forest site. Laccase gene types marked with ‘‘E’’ were also found expressed.
H. Kellner et al. / Soil Biology & Biochemistry 41 (2009) 1380–1389
observed in March, June 2004 and January 2005 (2.528–2.877)
(Table 3). The evenness of the fungal laccase gene population
reached values between 0.785 and 0.917, indicating an even
distribution of the sequenced clones among the different gene
types (Table 3). However, the mean richness and mean diversity
indices of the 3 soil replicates (plots) per sampling date (Table 3)
were not significant different among all sampling dates. The
highest dissimilarity between two consecutive samplings was
measured between October and January, representing a change of
more than 50% of the laccase genes present on these two
sampling dates (Fig. 4b). Dissimilarities among other sampling
dates ranged between 15 and 30% (Fig. 4b).
In a further step, the genetic difference between the laccase
gene populations was investigated. No significant difference
(UniFrac P Test and Arlequin FST; P < 0.05 significance level) of
the laccase gene populations obtained from genomic DNA
between different sampling dates was found (Table 4). The
clustering of the laccase gene populations amplified from soil
DNA showed no consistent trend between consecutive samplings,
except a clustering of the January and April 2005 populations
apart from the four other laccase gene populations obtained from
soil DNA extracts (Fig. 5).
1385
3.4. Expression of fungal laccase genes in soil samples
From RNA extracts that were processed in cDNAs, 18 and 33
different laccase gene types were amplified with the primer pair
combinations Cu1F/Cu2R and Cu1AF/Cu2R, respectively. As 9 gene
types were amplified with both primer pairs, altogether 42
different laccase genes were detected in the expression study. Gene
expression was observed at all sampling dates (Fig. 6a). The highest
relative expression rates, using both primer sets, were found in
August 2004 and January 2005 (Fig. 6a). The highest richness of
expressed genes was observed in January 2005 with 11 different
types per primer set, whereby some genes were revealed with both
primer sets. In August 2004, 11 different laccase genes were also
obtained but only with primer pair Cu1AF/Cu2R. A clearer picture of
the expression pattern could be drawn when analyzing the total
richness per sampling date, i.e. the total amount of expressed gene
types detected with both primer sets (‘‘S’’ in Fig. 6a). With 19
different expressed gene types, the highest richness was observed
in January 2005, followed by August and October 2004 (11), March
2004 (9), June 2004 (4) and April 2005 (2) (Fig. 6a). The attempt to
attribute the expressed genes to fungal groups and taxa via cluster
analysis (Fig. 2) showed expression shifts between asco- and
basidiomycetes. In January 2005, 16 of the expressed genes
belonged to basidio- and 3 to ascomycetes, whereas in October
2004, 7 different genes were expressed in asco- vs. 4 in basidiomycetes (Fig. 6a). The strong relative expression found in August
2004 corresponded to 7 different basidio- and 4 different ascomycete laccase genes (Fig. 6a).
Out of 28 expressed basidiomycete laccase genes, 13 (46%) could
be attributed to precise taxonomic levels, i.e. Agaricales sensu lato,
Atheliaceae, Sebacinaceae, B. nigrescens (Fig. 2). In January 2005, 7
Table 3
Number of sequences obtained from each single plot and plots combined for each
sampling date (community), analyzed for laccase gene richness (S), evenness (E) and
Shannon diversity index (H). Numbering of single soil cores followed sampling date
(March 2004–April 2005) and plot name (P2, P4, P6). Numbers in brackets for pooled
laccase gene richness include the abundances of each type found among three
possible plots.
Sampling analysis
Single plots
March 2004 P2
March 2004 P4
March 2004 P6
June 2004 P2
June 2004 P4
June 2004 P6
August 2004 P2
August 2004 P4
August 2004 P6
October 2004 P2
October 2004 P4
October 2004 P6
January 2005 P2
January 2005 P4
January 2005 P6
April 2005 P2
April 2005 P4
April 2005 P6
Fig. 4. Characterization of the basidiomycete laccase gene abundance separated by
putative nutritional status (a), and their change (dissimilarity) between two consecutive samplings according their presence and absence (b).
No. of sequences
22
21
25
17
23
20
24
26
25
23
27
26
21
24
19
23
25
17
Pooled data of 3 plots – ‘‘pooled community’’
March 2004
68
June 2004
60
August 2004
75
October 2004
76
January 2005
64
April 2005
65
Whole campaign
408
Laccase gene population
S
E
H
8
9
12
10
11
10
14
10
14
16
20
9
7
15
11
16
11
10
0.806
0.929
0.889
0.934
0.921
0.928
0.961
0.875
0.923
0.931
0.967
0.887
0.785
0.9
0.889
0.971
0.931
0.919
1.677
2.042
2.209
2.15
2.209
2.138
2.535
2.014
2.435
2.582
2.898
1.949
1.527
2.437
2.132
2.691
2.232
2.115
21 (29)
23 (31)
28 (38)
34 (45)
25 (33)
26 (37)
73
0.879
0.917
0.909
0.902
0.785
0.906
0.831
2.677
2.877
3.028
3.182
2.528
2.951
3.564
1386
H. Kellner et al. / Soil Biology & Biochemistry 41 (2009) 1380–1389
Table 4
Statistic significance (P values) of difference among the temporal soil DNA amplified laccase gene populations (DNA March 2004–April 2005) and the expressed laccase genes
(RNA all). Above diagonal, P values of UniFrac P Test and below diagonal, population differentiation based on 10,000 permutations (pairwise differences) using Arlequin. Bold
values indicate the significant different populations (P < 0.05).
DNA March 2004
DNA March 2004
DNA June 2004
DNA August 2004
DNA October 2004
DNA January 2005
DNA April 2005
RNA all
DNA June 2004
0.96
0.82
0.86
0.81
0.42
0.44