FEMS Microbiology Letters 247 (2005) 91–100
www.fems-microbiology.org
Analyses of soil fungal communities in adjacent natural forest
and hoop pine plantation ecosystems of subtropical Australia
using molecular approaches based on 18S rRNA genes
Jizheng He
a
a,b,*
,
Zhihong Xu a, Jane Hughes
a
Co-operative Research Centre (CRC) for Sustainable Production Forestry and Australian School of Environmental Studies,
Faculty of Environmental Sciences, Griffith University, Nathan, Qld. 4111, Australia
b
Research Centre for Eco-environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China
Received 4 January 2005; received in revised form 25 April 2005; accepted 25 April 2005
First published online 5 May 2005
Edited by E. Baggs
Abstract
Soil fungal communities were studied using 18S rDNA-based molecular techniques. Soil DNA was analyzed using temperature
gradient gel electrophoresis (TGGE), single-stranded conformational polymorphism (SSCP), cloning and sequencing methods, following community DNA extraction and polymerase chain reaction (PCR). The extracted community DNA was successfully amplified using the primer pair of EF4f-Fung5r which produced ca. 550 bp 18S rDNA fragments. TGGE screening of the PCR products
showed some differences in band position and intensity between two soil samples in adjacent natural forest (YNF) and hoop pine
plantation (YHP) ecosystems at Yarraman in subtropical Australia. TGGE and SSCP could be used for screening PCR products.
However, care must be exercised when interpreting the TGGE and SSCP results with respect to microbial diversity, because one
band may not necessarily represent one species. It is recommended that the PCR products should be purified before TGGE or SSCP
screening. SSCP screening of the clone sequences revealed differences among the clones. Sequence and phylogenetic analyses
revealed that all obtained clones were affiliated to the kingdom Fungi, including three phyla, i.e., Zygomycota, Ascomycota and
Basidiomycota. Our results suggested that community DNA extraction, PCR, cloning, SSCP screening of clones, sequencing of
selected clones and phylogentic analyses could be a good strategy in investigation of soil fungal community and diversity.
2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords: Soil fungal community; 18S rRNA gene; Cloning; TGGE; SSCP
1. Introduction
Fungi include organisms variously referred to as
moulds, mildews, rusts, smuts, yeasts, mushrooms and
puffballs. They play an important role in forest soil ecosystems in decomposing plant residues, promoting nutrient cycling and stimulating plant growth [1,2]. While
*
Corresponding author. Tel.: +86 10 6284 9788; fax: +86 10 6292
3563.
E-mail address: j.he@griffith.edu.au (J. He).
some fungi are well known to cause a range of plant diseases and in some cases to devastate crops, others are
known to antagonize plant pathogens. Some fungi
(external mycelium of arbuscular mycorrhizae) can also
affect the composition of bacterial communities, either
directly by changing host plant physiology, or indirectly
by changing the patterns of root exudation [3,4]. Knowledge of the structure and diversity of the fungal community in forest soil ecosystems is essential for sustainable
forest management and production. The composition,
distribution and diversity of soil fungal communities
0378-1097/$22.00 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.femsle.2005.04.033
92
J. He et al. / FEMS Microbiology Letters 247 (2005) 91–100
under different forest ecosystems may be useful as indicators of soil quality [5,6]. However, the fungal community in natural habitats is poorly known [7].
Soil microbial communities have been investigated
for many years using methods based on isolating and
culturing the microorganisms. Such techniques are
known for their selectivity and are not considered representative of the extent of the microbial community.
These cultivation-based methods such as soil dilution,
selective plating and enumeration of fungal propagules
using colony-forming units (CFU) have been routinely
applied for fungal identification and quantification.
While cultivation methods may be feasible for those fungi that grow rapidly or those that produce meiotic or mitotic propagules or fruiting bodies, many fungi at some
stages of their life cycle either lack distinctive traits, do
not sporulate, or have very specific growth requirements
which make these approaches impractical [8]. Moreover,
some fungal taxa such as the saprophytic basidiomycetes and the arbuscular endomycorrhiza, are difficult or
almost impossible to isolate from soil by dilution plating
[9]. Recent advances in the field of molecular biology
have made it possible to circumvent the cultivationbased problem. Culture-independent and DNA-based
molecular approaches offer a fast and sensitive alternative to the conventional cultivation techniques. They
are based on the analysis of single cells, offering an
opportunity to analyze the microbial community in its
full diversity. Numerous studies have applied these approaches to the study of soil microbial communities,
particularly of bacterial communities [6,10].
Only recently have several molecular biological approaches suitable for soil fungal community assessment,
such as 18S rDNA polymerase chain reaction-temperature gradient gel electrophoresis/denaturing gradient
gel electrophoresis (PCR-TGGE/DGGE) [11–13], cloning of rDNA [7,11,14], and real-time PCR analysis of
community DNA [8], been applied to elucidate fungal
community composition, diversity and quantification
in soils or plant roots. Smit et al. [11] designed various
PCR primers that allow the specific amplification of fungal 18S rDNA, even in the presence of nonfungal 18S
rDNA and therefore, greatly improved soil fungal community analyses based on 18S rDNA. They also successfully used the amplified rDNA for TGGE analysis and
produced reproducible and distinctive fingerprints for
rhizosphere and bulk soil samples. Gomes et al. [2]
investigated the fungal community dynamics in soil
and in the rhizosphere of two maize cultivars grown in
tropical soils using extracted DNA. A 1.65-kbp fragment of the 18S rDNA amplified from the total community DNA was analyzed by DGGE and by cloning and
sequencing. Pronounced changes in the composition of
fungal communities during plant growth development
were found [2]. The cloning and sequencing approach
provided information on the phylogeny of dominant
amplifiable fungal populations. Although these molecular approaches based on the analysis of 18S rRNA
genes are getting more and more widely used for the
assessment of fungal communities in soils e.g.,
[2,10,11,13,15,16], they have not yet been applied to
study the fungal community of soils in forest
ecosystems.
The aim of this work was to explore 18S rDNA-based
molecular approaches to investigate the soil fungal communities under contrasting natural forest and hoop pine
plantation ecosystems. Soil community DNA was extracted and PCR amplified. The PCR products were
then analyzed using TGGE, SSCP, cloning, and
sequencing methods.
2. Material and methods
2.1. Soil samples
The soil samples were collected from two contrasting
forest ecosystems located at a long-term experimental
site (2403 YMN, 2652 0 S, 15151 0 E) of Queensland
Department of Primary Industries – Forestry at Yarraman in subtropical Australia. Yarraman natural forest
(YNF) and the hoop pine (Araucaria cunninghamii)
plantation (YHP) sites are adjacent to each other on
the same position of the slope of approximately 2–3.
The YNF site is classified as a rainforest/bastard scrub
and dominated by bunya pine (Araucaria bidwilli), yellowwood (Terminalia oblongata), crows ash (Pentaceras
australis) and lignum-vitae (Premna lignum-vitae), with
emergent hoop pine. The YHP site was established after
clearing of the native forest in 1949. Under each forest
ecosystem, four areas of 10 · 10 m2 were selected and
five soil cores were taken from each area with an auger
(0–10 cm). Cores from the same ecosystem were well
mixed to form one soil sample. Each sample was placed
in a sterile plastic bag, sealed and transported to the laboratory in ice. All soils were passed through a 2.0-mm
sieve and stored at 4 C. The soil was classified as a
Snuffy Mesotrophic Red Ferrosol with a medium,
non-gravel, clay-loamy surface horizon [17]. Organic
matter contents were 10.9% and 11%, and pH values
were 5.79 and 5.47 for YNF and YHP samples,
respectively.
2.2. Soil DNA extraction, purification and PCR
amplification
DNA extraction was carried out using the modified
HolbenÕs method [18]. Briefly, duplicates of 5.0 g soil
samples (pre-washed with 0.1 M sodium phosphate)
were mixed by simple vortexing with 2.5 g large glass
beads (0.71–1.18 mm), 2.5 g small glass beads
(<0.11 mm) (SIGMA, acid washed), 0.15 g sodium
J. He et al. / FEMS Microbiology Letters 247 (2005) 91–100
dodecyl sulfate (SDS), and 15 ml of 1 mM sodium phosphate (pH 8.0). They were then incubated for 1 h in a
65 C water bath with end-over-end inversions every
10 min, shaken for 30 min at a speed of 250 cycles per
min, and centrifuged at 8000g for 15 min. The supernatants were incubated on ice for 30 min to precipitate
SDS and centrifuged at 8000g for 10 min at 10 C. The
cleared lysate (10 ml) was extracted with an equal volume of phenol–chloroform–isoamyl alcohol (25:24:1,
vol:vol:vol) and then with chloroform–isoamyl alcohol
(24:1, vol:vol). The aqueous phase was recovered by centrifugation and precipitated with 0.6 volume of cold isopropanol after freezing at 80 C for 1 h. The pellet of
crude DNA was obtained by centrifugation at 16,000g
for 20 min at 2 C, washed with cold 70% ethanol, and
suspended in TE buffer (10 mM Tris–HCl, 1 mM
EDTA, pH 8.0). The crude DNA was incubated for
15 min with 1/5 volume of 8 M potassium acetate on
ice followed by centrifugation at 13,800g, 4 C for
20 min. The supernatants were re-extracted with phenol–chloroform–isoamyl alcohol, precipitated with
isopropanol and re-suspended in EB buffer (10 mM
Tris–HCl, pH 8.5).
18S rDNA PCR amplification was carried out using
the fungal-specific primers, EF4f (5 0 -GGA AGG G[G/
A]T GTA TTT ATT AG-3 0 ) and Fung5r (5 0 -GTA
AAA GTC CTG GTT CCC-3 0 ) designed by Smit
et al. [11] and commercially synthesized (Sigma Genosys, Australia). The reaction mixtures were 50 ll which
usually contained 1· PCR buffer, 5 mM MgCl2, 2 mM
dNTPÕs, 2.5 U Taq polymerase from GibcoBRL,
10 lM each of primers and 1 ll (10 ng) DNA template. The thermal cycling scheme was heated to 94 C
for 3 min; then 35 cycles were run at 94 C for 1 min,
55 C for 1 min, and 72 C for 2 min; and finally 72 C
for 10 min.
2.3. TGGE and SSCP screening of the soil PCR products
TGGE screening of the PCR products (550 bp fragments) was conducted at 300 V with a temperature
range of 27–54 C on a QIAGEN TGGE-System (QIAGEN Gmbh, Hilden, Germany). The polyacrylamide gel
was composed of 5% (wt/vol) acrylamide, 0.17% (wt/vol)
bisacrylamide, 8 M urea, 2% (wt/vol) glycerol, 1· ME
buffer, 20 mM 3-[N-morpholino] propanesulfonic acid
(MOPS), 75 ll tetramethylethylenediamine (TEMED)
and 136 ll of 10% (wt/vol) ammonium persulfate
(APS) according to the manufacturerÕs instruction. The
gel was cast on to a gel support film (Gelbond PAG,
BMA, Rockland, MD) and polymerized for 60 min.
8 ll sample including 2 ll template DNA, 4 ll of 8 M
urea, 0.8 ll of 10· ME + dye buffer, and 1.2 ll H2O
were loaded into the gel and run in 1· ME buffer for
4 h. Gels were then silver-stained according to the manufacturerÕs protocol.
93
SSCP screening of the PCR products was conducted
at 300 V on a QIAGEN TGGE-System. One microlitre
of the PCR product was mixed with 4 ll formamide
loading dye (80% formamide, 10 mM EDTA, 0.1% xylene cyanol FF and 0.1% bromophenol blue, pH 8.0),
denatured at 95 C for 5 min and immediately chilled
on wet ice, then the previous denaturation step was repeated [19]. The denatured samples (8 ll) were then
loaded into the slots of the polyacrylamide gel and run
for 14 h at room temperature, and finally the gel was silver-stained. The polyacrylamide gel was composed of
8% (wt/vol) acrylamide, 0.21% (wt/vol) bisacrylamide,
5% (wt/vol) glycerol, 0.5 · TBE buffer (45 mM Tris–borate, 1 mM EDTA, pH 8.0), 93 ll TEMED and 232 ll
10% (wt/vol) APS, with a total volume of about
46.5 ml. The gel was cast on to a gel support film (Gelbond PAG, BMA, Rockland, MD) and polymerized at
least for 60 min.
2.4. Cloning of the PCR products
The PCR products were purified by 1.6% agarose gel
electrophoresis. The targeted band was excised and extracted using a QIAquick gel extraction kit (QIAGEN,
Australia). The cloning experiments were carried out
following the instructions of the manufacturer of the
pGEM-T Easy Vector System I (Promega, Madison,
WI). Briefly, 10 ng DNA was ligated into the plasmid
vector pGEM-T Easy Vector and transformed into competent cells (Escherichia coli JM109) by electroporation,
followed by the addition of 0.4 ml SOC buffer and incubation at 37 C for 1 h with agitation. Serial dilutions
were plated on to the LB (Luria–Bertani) plates with
ampicillin/IPTG/X-Gal and incubated overnight at
37 C. The white colonies in each plate were picked
out and cultured using LB broth overnight at 37 C with
agitation. The recombinant plasmid DNAs of the clone
cells were then extracted using QIAprep Spin Miniprep
Kits (QIAGEN, Australia) according to the instructions
of the manufacturer.
2.5. SSCP screening of the clones
The obtained clones were PCR re-amplified using the
primers of EF4f-Fung5r and the amplicons were purified using agarose gel electrophoresis. 1 ll of the PCR
product was mixed with 4 ll formamide loading dye,
denatured at 95 C for 5 min and immediately chilled
on wet ice. SSCP experiments were then conducted as
described in Section 2.3.
2.6. Sequencing of clones
The sequencing reactions of the selected clones were
performed using an ABI PRISM BigDye Terminator
Ready Reaction Kit according to the instructions of
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J. He et al. / FEMS Microbiology Letters 247 (2005) 91–100
the manufacturer (PE Applied Biosystems) with UPC/
M13 Reverse Sequencing Primer. A 10 ll reaction volume contained 4.0 ll Terminator Ready Reaction Mix
(PE), 3.2 ll primer (1 lM), 1.5 ll (40 ng) DNA template
and 1.3 ll H2O. All reagents were mixed and 25 cycles
were run with the following program: rapid thermal
ramp to 96 C and kept for 30 s; rapid thermal ramp
to 50 C and kept for 15 s; rapid thermal ramp to
60 C and kept for 4 min. The extension products were
purified by ethanol precipitation method and sequences
were determined on an ABI PRISM 377 Sequencer
(PE).
2.7. Sequence analyses and GenBank accession numbers
The clone sequences were manually proofread and
corrected if necessary, edited and aligned using BioEdit
and the associated ClustalW program [20]. The sequences were then analyzed with the NCBI Blast program and RDP Chimera Check program [21]. The
most similar GenBank sequences to the clones were extracted from the GenBank for including in the phylogenetic tree construction. Phylogenetic analyses were
conducted using MEGA version 2.1 [22] and a neighbor-joining (NJ) tree was constructed with 1000 replicates to produce Bootstrap values.
All the nucleotide sequences were submitted to Genbank and assigned accession numbers from AY576010
to AY576037.
community level physiological profiles [24]. How different storage would influence soil fungal communities remains unclear and deserves further investigation.
3.2. TGGE and SSCP screening of the soil PCR products
The PCR products were screened using TGGE techniques. As shown in Fig. 1, the TGGE gel separated the
PCR products into more than 10 different bands, and
there were some differences in band positions and intensities between the samples. For example, the five strongest bands (numbered in Fig. 1) for each sample were
slightly different. YNF had a strong band in position
3, but YHP had very weak bands at YNF position 3.
Moreover, at position 5, there were several strong
bands. In an attempt to determine the DNA sequences
of these bands, the 5 major representative bands numbered in Fig. 1 were excised and PCR was repeated using
the EF4f-Fung5r primer pair. The re-amplified TGGE
bands showed inconsistent product sizes on the agarose
gel, ranging from 300 to 550 bp (Fig. 2). The positions 1
and 2 PCR product bands were the correct size for the
soil DNA PCR fragments of 550 bp. However, when
the soil DNA PCR amplicons were purified using an
agarose gel before the TGGE experiments, only one major band was produced on the TGGE gel (result not
shown), which represented the band in position 2 in
3. Results and discussion
3.1. Soil DNA extraction and PCR amplification
The extracted soil community DNA was around
20 kbp in size (data not shown). The extracted DNA
was successfully amplified with fungal-specific primers
EF4f and Fung5r and produced products of ca.
550 bp, which was consistent with the results obtained
by Smit et al. [11,12]. Our initial DNA extraction experiments showed that the DNA extracted without beadbeating, e.g., method developed by Zhou et al. [23],
could not be successfully PCR amplified using 18S
rRNA primers, although it could be successfully amplified using 16S rRNA primers (data not shown). It appears that soil fungal DNA extraction requires some
method to achieve stronger lysis, such as the beadbeating. Moreover, fungal DNA extraction should be
conducted as soon as possible after sampling. The
DNA extraction experiments of this research were conducted within one week of sampling. However, we found
that there was no fungal PCR amplification for the soil
DNA extracts after about 4 months storage of the soil
samples at 4 C. Storage-induced changes in soil microbial communities have been observed using the Biolog
Fig. 1. Parallel TGGE gel of 18S rDNA PCR products (550 bp) of soil
samples from Yarraman natural forest (YNF) and hoop pine
plantation (YHP) with three replicated sample loadings. The gel was
run at 300 V for 4.0 h with a temperature gradient of 27–54 C. The
holes (1–5) showed the positions where bands were excised for further
study.
J. He et al. / FEMS Microbiology Letters 247 (2005) 91–100
Fig. 2. PCR amplification of the excised bands from the TGGE gel.
Nos. 1–5 indicated the positions of the bands in Fig. 1. PCR products at
positions 1 and 2 are ca. 550 bp; CK-negative control; L-100 bp ladder.
Fig. 1. This finding may suggest that caution should be
exercised in the interpretation of the TGGE gel results,
because some bands may not necessarily represent a real
microbial species.
SSCP also separated the PCR products into different
bands (data not shown). However, it was difficult to
judge the differences between the two treatments because
there were too many bands and the bands were not well
separated. There may be two reasons for this result [25].
One is re-annealing of samples after denaturation, which
can become critical when the high DNA concentrations
are used for community analyses. Re-annealing into
double-stranded DNA and the denatured singlestranded DNA can form more bands in the gel and thus
complicate the interpretation of the results. Another reason is the formation of heteroduplex DNA from PCR
products with similar sequences.
TGGE and SSCP both rely on electrophoretic separation based on differences in DNA sequences. They
were originally developed to detect point mutations in
DNA sequences and theoretically they can separate
DNA with one base-pair difference. However, owing
to the extreme complexity of soil microbial communities, these techniques can only separate the community
into several major bands (groups). It has been estimated
that DGGE/TGGE can only detect 1–2% of the microbial population representing dominant species present in
an environmental sample [26]. In addition, DNA fragments of different sequences may have similar mobilities
in the polyacrylamide gel. Therefore, a single band may
not necessarily represent a single species [27] and one
species may also give rise to multiple bands because of
multiple genes with slightly different sequences [27,28].
Maarit-Niemi et al. [28] used different combinations of
DNA extraction and purification procedures and found
that the method used influenced the banding pattern on
DGGE gels. Gelsomino et al. [27] found that direct and
indirect DNA extraction methods yielded DNA fingerprints that were 90% identical, with sample variation
for each extraction method being less than 5%. Most
of the differences in extraction methods and in reproducibility were between faint bands, presumably representing less dominant species [27]. DGGE/TGGE has been
used to assess the diversity of bacteria and fungi in the
95
rhizosphere, caused by changes in nutrient addition
and addition of anthropogenic chemicals [29]. The partial community level fingerprints derived from DGGE/
TGGE banding patterns have been analyzed for diversity studies based on the number and intensity of the
DNA bands as well as similarity between treatments.
However, with the limitations of PCR and of banding
pattern separation, care must be exercised when interpreting results with respect to microbial diversity. Different primer pairs may be needed to really cover the
fungal diversity in soil [30]. However, none of the above
studies indicated whether or not the PCR amplicons
were purified before using for TGGE/DGGE screening.
Specific DGGE/TGGE bands can also be excised from
gels, re-amplified and sequenced to provide more structural or functional diversity information. However, the
excised and re-amplified bands need to be checked on
an agarose gel to ensure that they are the appropriate
size. Moreover, prior to sequencing, the PCR products
should be cloned.
3.3. Clone and sequence analyses of the PCR products
Twenty-nine clones were obtained from the cloning
experiments, with 12 from YNF and 17 from YHP.
All these clones were re-amplified, and the obtained
PCR products (550 bp fragments) were purified on an
agarose gel and screened by SSCP. As shown in Fig.
3, most clone PCR products had one or two major
bands. There were differences among different clones,
although some of the differences were minor. For example, JH95 and JH96 just migrated to slightly different
positions. JH108 moved faster than all the others.
SSCP screening of the clone sequences suggested that
they were all unique and thus they all were sequenced.
Sequence results indicated that JH108 was only
434 bp, much shorter than others (ca. 550 bp), explaining why it moved faster than others on the SSCP gel.
The NCBI Blast search showed that there was no similar
Fig. 3. SSCP gel (300 V, 15 h) of 18S rDNA clone PCR products. The
PCR products were purified by agrose gel electrophoresis before using
for the SSCP screening. Nos. 88–100 belong to YNF and others to
YHP.
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J. He et al. / FEMS Microbiology Letters 247 (2005) 91–100
Table 1
Species of fungi with 18S rDNA sequences in the NCBI GenBank database most similar to the clones of Yarraman soil samples
Clone
no.
Fragment
size (bp)
Species with most
similar sequence
Taxon
% Identity
Accession no.
JH88
554
Mortierella hyalina
96.8
AY576010
JH89
558
Cyphellophora
laciniata
96.2
AY576011
JH90
561
Hydropisphaera
erubescens
97.0
AY576012
JH91
554
Penicillium
italicum
98.6
AY576013
JH92
552
Mortierella hyalina
95.6
AY576014
JH93
553
Mortierella
parvispora
97.0
AY576015
JH94
556
Mortierella
parvispora
95.9
AY576016
JH95
565
Calvatia gigantea
94.3
AY576017
JH96
555
Hyphodontia
alutaria
93.7
AY576018
JH97
559
Mortierella
parvispora
96.4
AY576019
JH98
560
Mortierella
parvispora
95.4
AY576020
JH100
557
Cladophialophora
boppii
95.7
AY576021
JH101
560
Mortierella hyalina
97.0
AY576022
JH102
558
Mortierella hyalina
96.8
AY576023
JH103
558
Mortierella hyalina
98.9
AY576024
JH104
560
Calvatia gigantea
94.9
AY576025
JH105
551
Mortierella hyalina
92.8
AY576026
JH106
562
Mortierella
parvispora
Zygomycota;
Zygomycetes;
Mortierellales
Ascomycota;
Pezizomycotina;
Chaetothyriomycetes;
Chaetothyriales
Ascomycota;
Pezizomycotina;
Sordariomycetes;
Hypocreomycetidae;
Hypocreales
Ascomycota;
Pezizomycotina;
Eurotiomycetes;
Eurotiales
Zygomycota;
Zygomycetes;
Mortierellales
Zygomycota;
Zygomycetes;
Mortierellales
Zygomycota;
Zygomycetes;
Mortierellales
Basidiomycota;
Hymenomycetes;
Homobasidiomycetes;
Lycoperdales
Basidiomycota;
Hymenomycetes;
Homobasidiomycetes;
Aphyllophorales
Zygomycota;
Zygomycetes;
Mortierellales
Zygomycota;
Zygomycetes;
Mortierellales
Ascomycota;
Pezizomycotina;
Chaetothyriomycetes;
Chaetothyriales
Zygomycota;
Zygomycetes;
Mortierellales
Zygomycota;
Zygomycetes;
Mortierellales
Zygomycota;
Zygomycetes;
Mortierellales
Basidiomycota;
Hymenomycetes;
Homobasidiomycetes;
Lycoperdales
Zygomycota;
Zygomycetes;
Mortierellales
Zygomycota;
Zygomycetes;
Mortierellales
93.7
AY576027
J. He et al. / FEMS Microbiology Letters 247 (2005) 91–100
97
Table 1 (continued )
Clone
no.
Fragment
size (bp)
Species with most
similar sequence
Taxon
% Identity
Accession no.
JH107
563
Mortierella
parvispora
96.0
AY576028
JH109
551
Mortierella hyalina
96.3
AY576029
JH111
559
Mortierella hyalina
91.1
AY576030
JH112
553
Mortierella
parvispora
95.2
AY576031
JH113
556
Mortierella hyalina
97.5
AY576032
JH114
555
Fabrella tsugae
96.6
AY576033
JH115
557
Mortierella hyalina
95.9
AY576034
JH116
560
Entoloma strictius
97.3
AY576035
JH117
552
Leptosphaeria
maculans
97.6
AY576036
JH118
553
Setosphaeria
monoceras
Zygomycota;
Zygomycetes;
Mortierellales
Zygomycota;
Zygomycetes;
Mortierellales
Zygomycota;
Zygomycetes;
Mortierellales
Zygomycota;
Zygomycetes;
Mortierellales
Zygomycota;
Zygomycetes;
Mortierellales
Ascomycota;
Pezizomycotina;
Leotiomycetes;
Helotiales
Zygomycota;
Zygomycetes;
Mortierellales
Basidiomycota;
Hymenomycetes;
Homobasidiomycetes;
Agaricales
Ascomycota;
Pezizomycotina;
Dothideomycetes;
Pleosporales
Ascomycota;
Pezizomycotina;
Dothideomycetes;
Pleosporales
94.8
AY576037
sequence in GenBank to this clone and it was removed
from all further analysis. Chimera check results indicated that no sequence was likely to be a chimera. The
most similar fungal species in the NCBI Genbank database to the Yarraman soil 18S rDNA clones are listed in
Table 1. There are 12 different species in the Genbank
which are most similar to the clone sequences, i.e., Mortierella hyaline and Mortierella parvispora (Zygomycetes, Zygomycota); Calvatia gigantean, Entoloma
strictius and Hyphodontia alutaria (Hymenomycetes,
Basidiomycota); and Fabrella tsugae, Penicillium italicum, Cyphellophora laciniata, Cladophialophora boppii,
Hydropisphaera erubescens, Leptosphaeria maculans
and Setosphaeria monoceras (Pezizomycotina, Ascomycota). In the RDP database, fungi were divided into five
phyla, i.e., Ascomycota, Basidiomycota, Zygomycota,
Chytriomycota and Fungi Incertae Sedis. In the NCBI
database, the fungi were divided into eight phyla, i.e.,
in addition to the five phyla of the RDP database,
Glomeromycota, Microsporidia and Unclassified fungi.
In fact, several sequences from the Yarraman fungal
clones were initially classified as unidentified fungi in
Genbank when compared with their best-matched se-
quences. These unidentified or uncultured fungi clones
were JH88, JH92, JH98, JH101, JH102, JH103,
JH111, JH113 and JH115. If a classification was assigned to these unidentified sequences based on subsequent better-matched Genbank sequences with
taxonomic definition, most of them could be relatives
of Mortierella hyaline or Mortierella parvispora species
belonging to the phylum Zygomycota.
The NJ tree of the clone sequences is shown in Fig. 4.
and their taxonomic assignments are listed in Table 2.
YNF clones were composed of 50% Zygomycota,
33.4% Ascomycota and 16.6% Basidiomycota. On the
other hand, YHP clones were composed of 68.7% Zygomycota, 18.8% Ascomycota and 12.5% Basidiomycota.
YNF had more Ascomycota but less Zygomycota clones
than that of YHP. These preliminary cloning and
sequencing results revealed that Zygomycota could be
the most important phylum of fungi in these forest soils.
The fungal community in the soil samples was distributed across three phyla, i.e., Zygomycota, Ascomycota
and Basidiomycota though only 28 clone sequences were
analyzed. In contrast, culture-based analyses of fungi
have identified mostly Ascomycetes [7,31,32].
98
J. He et al. / FEMS Microbiology Letters 247 (2005) 91–100
95
JH105, AY576026
Unidentified Zygomycota
JH111, AY576030
JH98, AY576020
JH112, AY576031
99
Unidentified Zygomycota
JH94, AY576016
92
JH97, AY576019
JH115, AY576034
77
JH93, AY576015
67
Unidentified Zygomycota
JH106, AY576027
71
JH107, AY576028
JH103, AY576024
Mortierella hyaline [AY157493]
Zygomycetes
Mortierella parvispora [AY129549]
55
94
JH102, AY576023
JH109, AY576029
JH101, AY576022
Unidentified Zygomycota
JH88, AY576010
JH92, AY576014
JH113, AY576032
JH96, AY576018
Unidentified Basidiomycota
JH104, AY576025
87
Hyphodontia alutaria [AF026615]
64
Calvatia gigantea [AF026622]
78
Hymenomycetes Basidiomycota
JH95, AY576017
76
70
JH116, AY576035
89
JH114, AY576033
93
JH117, AY576036
Fabrella tsugae [AF106015]
JH118, AY576037
94
JH89, AY576011
99
Cyphellophora laciniata [AY342010]
99
Pezizomycotina As comycota
JH100, AY576021
98
100
Cladophialophora boppii [AJ232946]
JH90, AY576012
Hydropisphaera erubescens [AY545722]
JH91, AY576013
99
Penicillium italicum [AF548091]
Fig. 4. Phylogenetic tree of Yarraman 18S rDNA clones (clone name, accession no.) and their most similar GenBank species (species name [accession
no.]). Numbers at branches are bootstrap values of 1000 replications. Phyla names of different groups of clones are assigned based on the
relationships of the clones to the known GenBank species and the NCBI Fungi Taxonomy classification. The tree is constructed using MEGA
software with the neighbor-joining method.
Table 2
Taxonomic compositions of 18S rDNA clones of Yarraman soil
samples as assigned from the NJ tree clusters
Taxonomic group
YNF
%
YHP
%
Zygomycota
Basidiomycota
Ascomycota
Total
6
2
4
12
50.0
16.7
33.3
100.0
11
2
3
16
68.7
12.5
18.8
100.0
The results of SSCP screening and sequencing of the
clones were consistent in that there were no identical
clones identified in the obtained 28 clones. This implied
that the fungal diversity in the soils could be very high.
As can be seen from Fig. 4, most clones belonging to the
phylum Ascomycota fitted into already described species. However, most clones belonging to Zygomycota
were unidentified. This could be due to more known
J. He et al. / FEMS Microbiology Letters 247 (2005) 91–100
Ascomycetes in the GenBank because they were culturable. The unidentified fungal sequences in the GenBank
may indicate some unknown groups of fungi in the soils.
This raises the possibility of the presence of novel
groups of fungi in these soils. More intensive sampling
and sequencing experiments are needed to really describe the fungal communities in the two soils.
One difficulty in some studies of fungal communities
based on 18S rDNA has been the selection of fungalspecific primers. To date the assessment of fungal diversity in natural systems by molecular means has been
hampered by the lack of sufficiently specific primers
[16]. White et al. [33,34] described a number of ÔuniversalÕ eukaryotic primers, which also amplify the fungi.
However, none of these primers is specific for fungi,
and their use for fungal community analysis in soil or
rhizosphere can only be recommended for systems with
low eukaryotic biodiversity, in which extensive controls
with respect to the nature of the bands obtained are required. The fungal-specific primers (EF4f and Fung5r)
used in this study were selected to provide optimal specificity for a wide range of fungi, theoretically excluding
amplification of virtually any other eukaryotic DNA
present in soil and rhizosphere [11]. Our study showed
that primer pair EF4f/Fung5r amplified target gene in
three major fungal phyla, yet did not produce amplicons
from plant, nematodes or bacteria.
In conclusion, this study provided possible strategies
for soil fungal community studies, i.e., community DNA
extraction, PCR amplification, cloning, SSCP screening
of clones, sequencing of selected clones and phylogentic
analyses to determine the taxonomic composition of the
community. TGGE and SSCP could be used for screening PCR products. However, care must be exercised
when interpreting the TGGE and SSCP results with respect to microbial diversity, because one band may not
necessarily represent one species. It is recommended that
the PCR products should be purified before using for
the TGGE or SSCP screening. Our preliminary results
using these molecular strategies have revealed some differences of fungal community composition between the
soil samples from the natural forest and the hoop pine
plantation. Three fungal phyla, i.e., Zygomycota, Ascomycota and Basidiomycota were detected from the YNF
and YHP samples. YNF appeared to have more Ascomycota but less Zygomycota than YHP, and most
clones in the phylum of Zygomycota are unidentified
species.
Acknowledgements
The funding support from Griffith University, Cooperative Research Centre for Sustainable Production Forestry, Australian Research Council, and Queensland
Department of Primary Industries – Forestry, is
99
acknowledged. We are grateful to Jing Ma for her technical assistance.
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