Botanical Studies (2009) 50: 57-68.
*
Corresponding author: E-mail: ssyang@ntu.edu.tw; Te l:
+886-2-33664456; Fax: +886-2-23679827.
INTRODUCTION
Soil microbes are essential components of the biotic
community in natural forests and are largely responsible
for ecosystem functioning (Hackl et al., 2004). The
microbial composition of the soil surface horizon has been
far better studied than that of the deeper horizons (Agnelli
et al., 2004). Microbes in the deeper horizons also play an
important role in ecosystem biogeochemistry (Madsen,
1995). It is not clear whether the subsurface microbial
community is closely related to the surface microbial
community or is an independent ecosystem with a distinct
assemblage of microorganisms (Fierer et al., 2003).
About 1% of the total number of microbes present in
soil is culturable (Schoenborn et al., 2004), hindering
analysis of microbial diversity using culture-based
methods. Various biochemical and molecular techniques
have been used to more completely and precisely
characterize microbes from the natural environment (Liu et
al., 2006). Although every method has its advantages and
limitations, 16S rRNA gene-based molecular techniques
have commonly been used to analyze the phylogenetic
diversity of bacterial communities (Chow et al., 2002).
Polymerase chain reaction (PCR) amplification of 16S
rDNA followed by separation of the PCR products on
a denaturing gradient gel electrophoresis (DGGE) is an
important method for analysis of bacterial communities
(Muyzer et al., 1993). Bacterial species can be identified
by generation of 16S rDNA clone libraries followed
by sequencing and comparison with databases of
ribosomal sequences, enabling phylogenetic affiliation to
cultured and uncultured microorganisms (Maidak et al.,
1999). These techniques have proven very suitable for
comparative fingerprinting of soil samples (Watanabe et
al., 2004).
A number of studies have shown that even small-
scale topographical landforms can alter environmental
conditions, which in turn retard or accelerate the activity
of organisms (Scowcroft et al., 2000). The effects
of topographical landforms on species composition,
productivity, environmental conditions, and soil
characteristics have been well investigated (Barnes et al.,
1998), but very few studies have investigated the effects
of these different environmental conditions on microbial
diversity.
The Fushan forest is one of the four natural forest sites
in the Taiwan Long Term Ecological Research Network
(TERN) to study the effect of environmental disturbances
such as typhoon and acidic deposition on ecosystem
function (Lin et al., 2000; Lin et al., 2003b; King et al.,
2003; Liu et al., 2004). However, a few studies have been
Soil bacterial community composition across different
topographic sites characterized by 16S rRNA gene
clones in the Fushan Forest of Taiwan
Shu-Hsien TSAI
1
, Ammaiyappan SELVAM
1
, Yu-Ping CHANG
1
, and Shang-Shyng YANG
1,2,
*
1
Institute of Microbiology and Biochemistry, and
2
Department of Biochemical Science and Technology, National Taiwan
University, Taipei 10617, Taiwan
(Received February 5, 2008; Accepted August 15, 2008)
ABSTRACT.
Bacterial communities present in soils from the valley, middle-slope, and ridge sites of the
Fushan forest in Taiwan were characterized using 16S rDNA analysis of genomic DNA after polymerase
chain reaction amplification, cloning, and denaturing gradient gel electrophoresis analysis. Phylogenetic
analysis revealed that the clones from nine clone libraries included members of the phyla Proteobacteria,
Acidobacteria, Actinobacteria, Bacteroidetes, Cyanobacteria, Firmicutes, Gemmatimonadetes, Nitrospirae,
Planctomycetes, candidate division TM7, and Verrucomicrobia. Members of Proteobacteria, Acidobacteria, and
Actinobacteria constituted 49.1%, 32.3%, and 6.3% of the clone libraries, respectively, while the remaining
bacterial divisions each comprised less than 6%. The ridge site exhibited the most bacterial species number,
indicating the influence of topography. Bacterial composition was more diverse in the organic layer than in the
deeper horizons. In addition, bacterial species numbers varied across the gradient horizons.
Keywords: 16S rDNA library; Acidobacteria; Bacterial community; DGGE; Proteobacteria; Topography.
mICROBIOlOgy
pg_0002
58
Botanical Studies, Vol. 50, 2009
conducted with Fushan forest soils: N mineralization and
nitrification rates (Owen et al., 2003), fluvial transportation
and sedimentation (Jen et al., 2006), and microbial
diversity (Tsai et al., 2007). In the present study, clone
libraries of 16S rDNA amplified fragments were used to
analyze the composition of bacterial communities from
three topographic sites in the Fushan forest. In addition,
soil characteristics, environmental conditions, and
bacterial diversity were compared in order to investigate
topographic effects on bacterial diversity.
mATERIAlS AND mETHODS
Site description
Fushan forest, located in northern Taiwan (24¢X34¡¦ N,
121¢X34¡¦ E), has an elevation ranging between 400 m and
1,400 m and is a moist, subtropical, mixed evergreen/
hardwood broad-leaf forest with a flora of over 500
species. Plant species belonging to the families Lauraceae,
Fagaceae, and Theaceae are dominant in this forest. Mean
annual precipitation is approximately 3,990 mm (Owen et
al., 2003). According to the Keys of Soil Taxonomy, the
soils of Fushan forest belong to Hapludults, Dystrochrepts,
Udipsamments, and Udorthents (Lin et al., 1996). Soil
samples were collected from three locations; the valley,
middle-slope, and ridge, which differed from each
other in altitude, slope, characteristic plant species, and
chemical characteristics (Table 1). The ridge site has the
highest floral diversity and density (Lin et al., 2003a). The
diversity gradually decreases through the middle-slope
and valley habitats. The ridge site also has the greatest
elevation, effective soil depth, and mean canopy height.
The middle-slope area has the highest slope, and the
valley has the greatest canopy gaps and the shallowest soil
formation, due to erosion.
Soil sampling
During September 2005, soil samples were collected
from the organic layer (above the topsoil, thickness
approximately 5-10 cm), topsoil (depth 0-20 cm), and
subsoil (depth 21-40 cm) in the valley, middle-slope, and
ridge. Three soil cores in each site were randomly collected
and separated into different soil layers. The samples
were sieved to 2 mm and each soil core was analyzed
separately for moisture content, pH, total organic carbon
(TOC), and total nitrogen (TN). Moisture content, pH, and
DNA extraction were done within 24 h of receiving the
samples; other properties were analyzed within two weeks
of sampling. Until the completion of all the analyses, the
soils were stored at 4¢XC. For DNA extraction, the same
layers in different cores of a site were pooled to yield a
composite sample, which was extracted at least thrice and
pooled for construction of the clone library. Air and soil
temperatures were measured with a thermometer directly
on site and under the soil at a depth of 5 cm, respectively.
Characteristics of the three tested sites and different layers
are shown in Table 1.
Chemical analyses
Moisture content was determined by drying the sample
at 105¢XC overnight to a constant weight. pH was measured
in 1:5 of soil: water extracts. Total organic carbon (TOC)
was determined using a modified Walkey-Black method, as
described by Nelson and Sommers (1982). Total nitrogen
(TN) was measured using a modified Kjeldahl method
(Yang et al., 1991). Chemical analyses were carried out
in triplicate, and the mean values and standard deviation
were expressed on a dry weight basis.
DNA extraction and purification
Genomic DNA of the soil samples was extracted from 2
g of fresh soil following a modified protocol of Krsek and
Wellington (1999) with Crombach buffer (33 mM Tris-
HCl, pH 8.0; 1 mM EDTA, pH 8.0) containing lysozyme
(5 mg ml
-1
) and sodium dodecyl sulfate (1%). After
centrifugation, supernatants were subjected to potassium
acetate and polyethylene glycol precipitation, phenol/
chloroform/iso-amylalcohol purification, isopropanol
precipitation, and spermine-HCl precipitation. The
crude DNA was purified using a Gene-Spin
TM
1-4-3
DNA Extraction Kit (Protech, Protech Technology
Enterprise Ltd, Taiwan) according to the manufacturer ¡¦
s recommendations and stored at -20¢XC. DNA extractions
were repeated to obtain at least three measurements in a
composite sample.
PCR amplification of 16S rDNA
Bacterial 16S rDNA was amplified by PCR
using the universal eubacterial primers 10f
(5¡¦- AGTTTGATCCTGGCTCAG-3¡¦) and 1507r
(5¡¦-TACCTTGTTACGACTTCA CCCCA-3¡¦). The
Escherichia coli numbering positions (in the 16S rDNA)
of the primers 10f and 1507r are 10-27 and 1507-1485,
respectively (Heyndrickx et al., 1996). The 50 £gl reaction
contained 25 pmol of each primer, 200 £gM of each dNTP
(Protech), 1¡Ñ PCR buffer (Protech, with MgCl
2
), 1.5 U of
Pro Taq DNA polymerase (Protech), and 1 £gl of DNA.
PCR was performed using an Applied Biosystems
2720 Thermal Cycler (Foster City, CA, USA ) with the
following reaction conditions: 94¢XC for 5 min, followed by
35 cycles at 95¢XC for 1 min, 55¢XC for 30 s, 72
o
C for 1 min,
and a final extension step at 72¢XC for 10 min. The PCR
products (5 £gl) were examined by electrophoresis on a 1¡Ñ
TAE agarose gel (2% w v
-1
) with a 100 bp DNA ladder
(Promega, Madison, WI, USA) as a marker to confirm the
size and approximate quantity of the generated amplicons.
Construction and analysis of clone libraries
The PCR products of the 16S rRNA genes were
completely loaded onto a 2% low melting agarose gel
(Invitrogen, San Diego, CA, USA). The band, with an
expected size of approximately 1,500 bp, was cut and
purified with a Gel Extraction Kit (Qiagen, CA, USA)
following the manufacturer¡¦s instructions and subsequently
pg_0003
TSAI et al. ¡X Bacterial community in Fushan Forest soils
59
ligated into pGEM-T Vector Systems (Promega). The
ligation product was transformed into competent E. coli
JM109 cells, and the clones were isolated by blue-white
screening with isopropyl-£]-D-thiogalactopyranoside
(IPTG, 0.2 mM) and 5-bromo-4-chloro-3-indolyl-£]-D-
galacto-pyranoside (X-Gal, 0.1 mM). White colonies were
plated on LB agar containing 100 £gg ml
-1
ampicillin. Each
plate contained approximately 100 clones, and 40 clones
were randomly selected to represent each of the nine
composite forest soil samples.
PCR screening of clone libraries, DggE, and
sequencing
PCR screening of 360 transformants was carried out
as described by Schabereitner-Gurtner et al. (2001). The
vector-specific forward primer T7 (5¡¦-TAA TAC GAC
TCA CTA TAG GG-3¡¦) and reverse primer SP6 (5¡¦-ATT
TAG GTG ACA CTA TAG AAT AC-3¡¦) were used in 25
£gl reaction mixture containing 2.5 £gl DNA extract as a
template. Three hundred and fifty positive transformants
were confirmed based on a length of approximately 1,500
Table 1. Some environmental characteristics and soil properties of the study sites in the Fushan forest.
Variables
Valley
Middle-slope
Ridge
Altitude (m)
700
850
1000
Characteristic plant species Aralia bipinnata,
Cinnamomum micranthum Ilex goshiensis, I. uraiensis,
Callicarpa dichotoma,
Itea parviflora, Myrsine sequinii,
Cyclobalanopsis gilva,
Rhododendron ellipticum,
Villebrunea pedunculata
Syzygium buzifolium,
Ternstroemia gymnanthera
Soil texture
Lithosols, stony loam
Colluviums, stony loam Yellow soils, stony loam
Air temperature (¢XC)
25.6
¡Ó
0.6
a
24.8¡Ó0.6
ab
24.1¡Ó0.7
b
Soil temperature (¢XC)
22.5¡Ó0.6
a
22.5¡Ó0.6
a
22.2¡Ó0.7
a
pH
Organic layer
4.4¡Ó0.1
a,B
4.3¡Ó0.1
a,B
4.3¡Ó0.1
a,B
Topsoil (0-20 cm)
4.7¡Ó0.1
a,A
4.6¡Ó0.1
a,A
4.6¡Ó0.1
a,A
Subsoil (21-40 cm)
4.8¡Ó0.1
a,A
4.7¡Ó0.2
a,A
4.7¡Ó0.1
a,A
Moisture content (g kg
-1
)
Organic layer
531.3¡Ó5.9
b,A
589.2¡Ó53.1
ab,A
614.4¡Ó26.2
a,A
Topsoil (0-20 cm)
453.4¡Ó51.5
a,B
495.2¡Ó32.4
a,B
520.8¡Ó2.3
a,B
Subsoil (21-40 cm)
347.4¡Ó12.3
b,C
364.0¡Ó50.1
b,C
497.8¡Ó8.1
a,B
Total organic carbon (g kg
-1
)
Organic layer
120.2¡Ó12.2
b,A
156.2¡Ó12.5
a,A
162.8¡Ó5.1
a,A
Topsoil (0-20 cm)
68.7¡Ó2.6
c,B
94.4¡Ó1.6
b,B
107.0¡Ó6.2
a,B
Subsoil (21-40 cm)
25.0¡Ó4.5
b,C
56.5¡Ó3.5
a,C
59.3¡Ó4.7
a,C
Total nitrogen (g kg
-1
)
Organic layer
4.8¡Ó0.5
b,A
7.0¡Ó0.5
a,A
7.7¡Ó0.2
a,A
Topsoil (0-20 cm)
3.0¡Ó0.1
b,B
4.8¡Ó0.5
a,B
5.4¡Ó0.4
a,B
Subsoil (21-40 cm)
1.1¡Ó0.1
b,C
3.3¡Ó0.3
a,C
3.5¡Ó0.6
a,C
C/N ratio
Organic layer
25.3¡Ó3.6
a,A
22.6¡Ó3.3
a,A
21.3¡Ó0.7
a,A
Topsoil (0-20 cm)
22.9¡Ó1.2
a,A
19.5¡Ó2.3
b,AB
19.8¡Ó0.4
b,A
Subsoil (21-40 cm)
22.7¡Ó2.9
a,A
17.1¡Ó1.2
b,B
16.9¡Ó1.6
b,B
Means¡ÓS.D (n = 3). Means in the same row that do not share the same lower case alphabetic superscript are significantly different
at the 5% level according to Duncan¡¦s multiple range test (DMRT). Means for a variable in the same column that do not share the
same upper case alphabetic superscript are significantly different at the 5% level according to DMRT. Air and soil temperature
data correspond to a single date and measured at the time of sampling.
pg_0004
60
Botanical Studies, Vol. 50, 2009
bp. The PCR products were again amplified using the
primer set GC clamp-968f (5¡¦-CGC CCG GGG CGC GCC
CCG GGC GGG GCG GGG GCA CGG GGG GAA CGC
GAA GAA CCT TA-3¡¦) and 1401r (5¡¦-GCG TGT GTA
CAA GAC CC-3¡¦) as nested PCR (Felske et al., 1997).
Reaction mixtures were the same as those described for
the 16S rDNA amplification. The PCR reaction conditions
were: 94¢XC for 90 s, followed by 33 cycles at 95¢XC for 20
s, 56¢XC for 30 s, 72¢XC for 45 s, and a final extension step
at 72¢XC for 10 min. PCR products, 5 £gl, were analyzed
by 2% (w v
-1
) agarose gel electrophoresis. The PCR
products (20 £gl) were separated at 60¢XC on a vertical
denaturing gradient gel using the Dcode
TM
Universal
Mutation Detection System (Bio-Rad Laboratories,
Hercules, CA, USA). Polyacrylamide (6%) gels with
gradients of 45-65% denaturants (where 100% denaturants
contained 7 M urea and 40% formamide) were prepared
in accordance with Muyzer et al. (1996), and a running
time of 6 h at 150 V was selected as these conditions
optimally separated the maximal number of bands. After
electrophoresis, gels were stained with ethidium bromide
(0.5 £gg ml
-1
) and photographed under UV light. The inserts
of clones showing different positions in the DGGE were
subsequently sequenced (Mission Biotech., Taiwan) using
the primers SP6 and T7.
Statistical analysis and sequence and
phylogenetic analyses
Differences in the physico-chemical variables due to
site and soil depth were tested by the analysis of variance
and Duncan¡¦s multiple range test with SPSS version 11.5
software. Significance was accepted at p level < 0.05.
The close relatives and phylogenetic affiliation of the
sequences obtained were checked using the Basic Local
Alignment Search Tools (BLAST) search program at the
National Center for Biotechnology Information (NCBI)
website (http://www.ncbi.nlm.nih.gov/). Homology
search (GenBank/EMBL/DDBJ) was performed using the
BLAST program (Altschul et al., 1990) in the reference
database. The sequences were also submitted to the
Chimera Check program of Ribosomal Database Program
(RDP) to identify chimeric artifacts. In order to obtain
the rarefaction curves for the three sampling sites, species
richness was plotted using BioDiversity Pro version 2, as
instructed by the software manual (http://www.sams.ac.uk/
dml/projects/benthic/bdpro/downloads.htm). To determine
species diversity, the number of species was plotted against
the number of individuals, a steeper curve indicating more
diverse communities in the sample. This plot is robust
against sample size effects, enabling comparisons to be
made across sampling sites (Simberloff, 1972).
Nucleotide accession number
The 16S rDNA sequence data for the clones reported
in this article have been submitted to the NCBI nucleotide
sequence database under the accession numbers
DQ451440-DQ451528.
RESUlTS
Soil properties and environmental conditions
The physico-chemical characteristics of the soil in the
valley, middle-slope, and ridge sites are shown in Table 1.
The Fushan forest soil is stony loam, and the soil texture
is lithosol in the valley, colluvium in the middle-slope, and
yellow soil in the ridge. The soils were acidic (pH 4.3-4.8),
and the pH gradually increased through the deeper layers;
there were significant pH differences between the organic
layer and topsoil or subsoil (p<0.05). The valley samples
had the highest pH, but the pH differences among the
sites were not significant (p>0.05). The TOC and TN
contents of the soils were significantly higher (p<0.05) in
the middle-slope and the ridge than the valley. The soil
moisture content was the significantly highest in the ridge
(p<0.05) among the three tested sites; while the TOC, TN,
and C/N ratio were the significantly highest (p<0.05) in
the organic layer among the three tested depths.
Analyses of clone libraries
Of the 350 clones analyzed by DGGE, 89 unique
sequences were identified in the nine clone libraries
(Table 2). Clones were members of 11 bacterial
phyla: Proteobacteria, Acidobacteria, Actinobacteria,
Bacteroidetes, Cyanobacteria, Firmicutes,
Gemmatimonadetes, Nitrospirae, Planctomycetes,
candidate division TM7, and Verrucomicrobia. Members
of Proteobacteria (49.1%) and Acidobacteria (32.3%)
dominated the clone libraries. Within the phylum
Proteobacteria, £\- and £^-Proteobacteria were the most
numerous (16.6% and 15.1%, respectively), followed by
£]- and £_-Proteobacteria. Actinobacteria constituted 6.3%
of the clone library, and each of the remaining bacterial
divisions constituted < 6% of the clone library (Figures 1
and 2).
Differences in bacterial composition across
sampling sites
Rarefaction curves for the three sampling sites are
Figure 1. Phylogenetic affiliation of 16S rDNA clones (n = 350)
from three soil sampling sites.
pg_0005
TSAI et al. ¡X Bacterial community in Fushan Forest soils
61
Table 2. Identification of bacterial strains in 16S rDNA clone library isolated from soils of Fushan forest.
Clone Closest related organism in the database
Accession number
of reference strain Identity (%)
Taxon
FAC1 Acidobacteria bacterium Ellin7137
AY673303 96 (1387/1438) Acidobacteria
FAC2 Acidobacteria bacterium Ellin7184
AY673350 96 (1410/1456) Acidobacteria
FAC3 Uncultured Acidobacteria bacterium clone JG36-GS-146
AJ582043 97 (1412/1454) Acidobacteria
FAC4 Uncultured Acidobacteria bacterium clone AKYH694
AY922148 96 (952/986) Acidobacteria
FAC5 Uncultured Acidobacteria bacterium clone EB1071
AY395390 95 (1173/1224) Acidobacteria
FAC6 Uncultured Acidobacteria bacterium clone EB1129
AY395448 98 (1369/1390) Acidobacteria
FAC7 Uncultured Acidobacteria bacterium clone JG36-GS-146
AJ582043 96 (1400/1453) Acidobacteria
FAC8 Uncultured Acidobacterium UA3
AF200699 98 (1441/1458) Acidobacteria
FAC9 Uncultured Acidobacterium UA3
AF200699 97 (1428/1458) Acidobacteria
FAC10 Uncultured Holophaga sp. clone JG37-AG-54
AJ519374 97 (1225/1256) Acidobacteria
FAC46 Uncultured Holophaga sp. clone JG30a-KF-86
AJ536874 94 (1207/1279) Acidobacteria
FAC52 Uncultured Holophaga sp. clone JG37-AG-31
AJ519368 99 (1320/1333) Acidobacteria
FAC53 Uncultured Acidobacteria bacterium clone AKYG469
AY922023 96 (1359/1405) Acidobacteria
FAC57 Uncultured Acidobacteria bacterium clone VHS-B3-48
DQ394942 94 (1415/1494) Acidobacteria
FAC59 Uncultured Acidobacteria bacterium clone EB1071
AY395390 96 (1359/1407) Acidobacteria
FAC60 Uncultured Acidobacteria bacterium clone FTL227
AF529104 94 (1325/1403) Acidobacteria
FAC62 Uncultured Acidobacteria bacterium G03_WMSP2
DQ450698 95 (1122/1174) Acidobacteria
FAC65 Uncultured Acidobacteria bacterium F04_WMSP1
DQ450716 95 (1299/1363) Acidobacteria
FAC66 Uncultured Acidobacteria bacterium JG36-GS-126
AJ582044 94 (1376/1453) Acidobacteria
FAC70 Uncultured Acidobacteria bacterium clone B08_WMSP1
DQ450707 93 (1261/1423) Acidobacteria
FAC71 Uncultured Holophaga sp. clone JG30-KF-C37
AJ536864 98 (1370/1394) Acidobacteria
FAC72 Uncultured soil baterium DUNssu164
AY913371 97 (1433/1466) Acidobacteria
FAC77 Uncultured Acidobacteria bacterium clone C10_WMSP1
DQ450710 98 (1332/1356) Acidobacteria
FAC80 Uncultured Holophaga sp. clone JG37-AG-61
AJ519377 94 (1202/1278) Acidobacteria
FAC86 Bacterium Ellin5017
AY234434 97 (1354/1386) Acidobacteria
FAC88 Uncultured Acidobacteria bacterium DOK_NOFERT_clone590
DQ829504 91 (479/522) Acidobacteria
FAC11 Bacterium Ellin332
AF498714 99 (1396/1405) £\-Proteobacteria
FAC12 Bacterium Ellin6089
AY234741 96 (1394/1441) £\-Proteobacteria
FAC13 Methylocapsa acidiphila
AJ278726 96 (1360/1415) £\-Proteobacteria
FAC14 Ochrobactrum sp. ¡¥Relman 1999¡¦
AF028733 88 (823/928) £\-Proteobacteria
FAC15 Stella vacuolata DSM5901
AJ535711 92 (1323/1434) £\-Proteobacteria
FAC16 Uncultured Alpha-proteobacterium JG37-AG-26
AJ518768 96 (1218/1265) £\-Proteobacteria
FAC17 Uncultured bacterium FukuS110
AJ289986 93 (1348/1443) £\-Proteobacteria
FAC49 Uncultured Alphaproteobacterium F12_WMSP1
DQ450764 93 (1265/1355) £\-Proteobacteria
FAC58 Acidosphaera rubrifaciens
D86512
94 (1372/1450) £\-Proteobacteria
FAC67 Uncultured Alphaproteobacterium JG30-KF-C3
AJ536857 92 (843/908) £\-Proteobacteria
FAC68 Uncultured type II methanotroph clone 18
AY163571 95 (1307/1364) £\-Proteobacteria
FAC69 Uncultured Alphaproteobacterium clone D05_WMSP1
DQ450762 96 (905/940) £\-Proteobacteria
FAC73 Uncultured Alphaproteobacterium clone EB1033
AY395352 93 (1284/1378) £\-Proteobacteria
FAC82 Uncultured Alphaproteobacterium clone EB1127
AY395446 97 (1355/1393) £\-Proteobacteria
FAC83 Alphaproteobacterium Shinshu-th1
AB121772 96 (1397/1445) £\-Proteobacteria
FAC18 Acidovorax sp. KSP2
AB076843 97 (1445/1485) £]-Proteobacteria
FAC19 Burkholderia sp. TNFYE-5
AF508806 97 (1446/1489) £]-Proteobacteria
FAC20 Delftia sp. LFJ11-1
DQ140182 99 (1487/1490) £]-Proteobacteria
FAC21 Uncultured bacterium MS8
AF232922 96 (997/1032) £]-Proteobacteria
pg_0006
62
Botanical Studies, Vol. 50, 2009
Table 2. (Continued)
Clone Closest related organism in the database
Accession number
of reference strain Identity (%)
Taxon
FAC22 Uncultured Green Bay Ferromanganous micronodule bacterium
MNC9
AF293007 96 (1438/1490) £]-Proteobacteria
FAC43 Variovorax sp. KS2D-23
AB196432 96 (1432/1491) £]-Proteobacteria
FAC44 Uncultured bacterium MS8
AF232922 95 (1357/1418) £]-Proteobacteria
FAC45 Uncultured bacterium clone B44
AF407722 96 (1369/1418) £]-Proteobacteria
FAC51 Burkholderia sp. isolate N3P2
U37344
96 (1442/1487) £]-Proteobacteria
FAC61 Uncultured Betaproteobacterium clone F03_Pitesti
DQ378169 97 (1444/1487) £]-Proteobacteria
FAC64 Uncultured Betaproteobacterium clone F03_Pitesti
DQ378169 96 (1439/1487) £]-Proteobacteria
FAC23 Dyella japonica XD53
AB110498 99 (1468/1481) £^-Proteobacteria
FAC24 Legionella-like amoebal pathogen HT99
AY741401 96 (1426/1485) £^-Proteobacteria
FAC25 Pantoea agglomerans ChDC YP1
AY691543 98 (1481/1496) £^-Proteobacteria
FAC26 Pseudomonas sp. NZ096
AY014817 99 (1391/1403) £^-Proteobacteria
FAC27 Uncultured Alteromonadales bacterium clone BL011B19
AY806128 92 (682/704) £^-Proteobacteria
FAC28 Uncultured Gammaproteobacterium clone BIfciii1
AJ318123 92 (953/1027) £^-Proteobacteria
FAC29 Uncultured Gammaproteobacterium YNPRH65B
AF465652 94 (1386/1460) £^-Proteobacteria
FAC42 Pantoea agglomerans WAB1927
AM184266 99 (1479/1486) £^-Proteobacteria
FAC50 Xanthomonadaceae bacterium Ellin7015
AY673181 97 (1399/1434) £^-Proteobacteria
FAC63 Uncultured Gammaproteobacterium 308
AB252888 91 (1296/1409) £^-Proteobacteria
FAC76 Dyella koreensis strain BB4
AY884571 98 (1231/1253) £^- Proteobacteria
FAC81 Uncultured Xanthomonas sp. clone TM17_46
DQ279336 91 (1377/1497) £^- Proteobacteria
FAC41 Uncultured Deltaproteobacterium clone EB1076
AY395395 93 (1376/1474) £_-Proteobacteria
FAC48 Uncultured Deltaproteobacterium clone BSR2LA02
AY690092 90 (750/828) £_-Proteobacteria
FAC55 Uncultured Deltaproteobacterium clone BPM3_B01
AY689889 96 (786/816) £_-Proteobacteria
FAC56 Uncultured Deltaproteobacterium clone AKYH1423
AY921676 95 (1341/1397) £_-Proteobacteria
FAC79 Uncultured Entotheonella sp. clone Dd-Ent-69
AY897120 92 (949/1031) £_-Proteobacteria
FAC87 Uncultured Deltaproteobacterium clone KY221
AB116509 92 (904/975) £_-Proteobacteria
FAC30 Uncultured actinobacterium Elev_16S_853
EF019692 99 (1351/1361) Actinobacteria
FAC31 Uncultured actinobacterium Amb_16S_1709
EF019097 98 (1349/1363) Actinobacteria
FAC74 Uncultured Actinobacterium clone CrystalBog1D10
AY792234 94 (1317/1389) Actinobacteria
FAC75 Uncultured Actinobacterium clone CrystalBog1C4
AY792233 94 (1310/1393) Actinobacteria
FAC78 Frankia sp. symbiont in root nodule FE37
AF063641 92 (1348/1457) Actinobacteria
FAC32 Uncultured Bacteroidetes bacterium clone BIti15
AJ318185 94 (878/931) Bacteroidetes
FAC33 Uncultured Bacteroidetes bacterium clone SW30
AJ575720 95 (1402/1470) Bacteroidetes
FAC54 Uncultured Flexibacter sp. clone TM19_36
DQ279370 91 (1158/1265) Bacteroidetes
FAC34 Cylindrospermum sp. PCC 7417
AJ133163 92 (1320/1433) Cyanobacteria
FAC35 Bacillus weihenstephanensis
AB021199 99 (1495/1504) Firmicutes
FAC36 Veillonella parvula strain ATCC 17745
AY995769 99 (1490/1494) Firmicutes
FAC40 Bacillaceae bacterium KVD-1700-08
DQ490381 99 (1470/1477) Firmicutes
FAC37 Uncultured Gemmatimonadetes bacterium clone EB1081
AY395400 98 (985/995) Gemmatimonadetes
FAC38 Uncultured Green Bay Ferromanganous micronodule bacterium
MNC2
AF293010 96 (1444/1495) Nitrospirae
FAC39 Uncultured bacterium SBR2013
AF269000 91 (1221/1329)
TM7
FAC47 Isophaera sp.
X81958
95 (1209/1272) Planctomycetes
FAC84 Uncultured Verrucomicrobia subdivision 3 bacterium clone EB1106 AY395425 95 (1400/1463) Verrucomicrobia
FAC89 Verrucomicrobia bacterium clone B-E3
DQ516404 97 (539/555) Verrucomicrobia
FAC85 Uncultured soil bacterium clone C062
AF507696 96 (1361/1405) Unclassified
pg_0007
TSAI et al. ¡X Bacterial community in Fushan Forest soils
63
shown in Figure 3. The ridge site exhibited a more diverse
microbial composition than the middle-slope or valley
site. Cluster analysis (Figure 4) showed that the middle-
slope and ridge sites clustered together (similarity) with
a Jaccard index (Ji) of 22.7%, and the valley site formed
another cluster sharing 19.4% Ji with the other two
sites. Among different soil layers, topsoil and subsoil
cluster together, leaving the organic layer as a separate
cluster in the middle-slope and ridge sites. However,
in the valley, the organic layer and topsoil clustered
together. Proteobacteria, Acidobacteria, Actinobacteria,
and Firmicutes were evenly distributed across the three
sites, as indicated by the exact number of clones. At
all three sampling sites, Proteobacteria were dominant.
Gemmatimonadetes and Nitrospirae were only observed
in the organic layer of the valley. Planctomycetes and
Verrucomicrobia were found only in the organic layer
of the middle-slope and ridge while Cyanobacteria were
Figure 2. Phylogenetic relationship of Fushan forest clone based
on partial 16S rDNA gene sequences with 16S rDNA reference
gene sequences available in NCBI. Reference sequences were
determined after BLAST research. The tree has been rooted with
the archaeal 16S rDNA sequence from Archaeoglobus fulgidus.
The scale-bar indicated the substitution rate per nucleotide site.
(a) Acidobacteria; (b) Alphaproteobacteria; (c) Betaproteobacte-
ria; (d) Gammaproteobacteria, and (e) Deltaproteobacteria.
pg_0008
64
Botanical Studies, Vol. 50, 2009
found in the organic layer of ridge. The candidate division
TM7 was only found in the subsoil of the middle-slope
(Table 3).
Differences in bacterial composition across soil
depths
The relative abundance of specific microbial groups at
the three soil depths are shown in Table 3. The bacterial
composition was the most diverse in the organic layer.
The reduction in number of bacterial species with
progressive depth was greater at the ridge site than in the
middle-slope or valley site. For the ridge site, topsoil and
subsoil exhibited 25.0% and 40.6% reductions in the total
species number when compared to the organic layer. The
reductions in number of bacterial species in the valley of
topsoil and subsoil were 14.8% and 33.3%, respectively,
and the middle-slope area had values of 16% and 24%.
The proportional abundance of Gram-positive bacteria
(Acidobacteria and Actinobacteria) increased with soil
depth while the proportional abundance of Gram-negative
bacteria (Proteobacteria) decreased with soil depth.
DISCUSSION
Soil at Fushan forest was acidic (pH 4.3-4.8), and the
pH gradually increased through the deeper layers, as has
been reported for the spruce, hemlock and grassland soils
of Tatachia forest in Taiwan (Yang et al., 2003, 2006;
Cho et al., 2008). Of the three sampling sites, the valley
samples had the highest pH due to the lowest amount
of organic matter, which was a result of large canopy
gaps and consequently low leaf littering. However, the
differences were not significant (p>0.05). The TOC and
TN contents of the soil were the highest in the ridge,
followed by the middle-slope, and those in the valley were
the lowest, which correlates with the floral density. Lin et
al. (2003a) reported that the biomass of woody debris was
higher in the ridge (36.1 Mg ha
-1
) than in the valley (8.5
Mg ha
-1
).
Acidobacteria and Proteobacteria are generally the
most numerically dominant phyla in soil while members
of Bacteroidetes and Firmicutes are less common (Dunbar
et al., 1999; Chow et al., 2002; Fierer et al., 2005). In this
study, we also found Proteobacteria and Acidobacteria
to be dominant. Members of £\-Proteobacteria were also
found to be the most abundant in the 16S rDNA clone
libraries derived from Long-Term Soil Productivity (LTSP)
forest soil from British Columbia, Canada (Chow et al.,
2002), Australian forest soils (Stackebrandt et al., 1993),
Scotland grassland rhizosphere soil (McCaig et al., 1999),
and fertilizer-applied soil (Toyota and Kuninaga, 2006).
Members of Acidobacteria were the most abundant in the
clone libraries from Arizona pinyon pine rhizosphere and
bulk soils (Dunbar et al., 1999), and in desert, prairie,
and forest soils (Fierer et al., 2005). The representation
of phyla in this study is very similar to that reported by
Kraigher et al. (2006) using a clone library from fen soil
with high organic carbon content (150 g kg
-1
). Among the
Proteobacteria, £\-Proteobacteria were the most prevalent,
followed by £]-Proteobacteria and £^-Proteobacteria
(Figure 2). Similarly, the proportions of Actinobacteria,
Bacteriodetes, and Firmicutes in the clone library of this
study (4.6%, 2.6%, and 3.8%, respectively) were similar
to those reported by Kraigher et al. (2006) (6.1%, 3.5%,
and 2.6%, respectively). The Fushan soils also had high
levels of TOC (25.0-162.8 g kg
-1
), implying the influence
of organic carbon in determining bacterial diversity. The
relative abundance of Actinobacteria was substantially
lower in the rDNA clone library than in clone libraries
from mineral soil of Williams Lake LTSP plots (Axelrood
et al., 2002) and Neocaledonian mine spoils (Hery et al.,
2005). Krave et al. (2002) also reported very low (1.4%)
representation of Acidobacteria in a clone library of litter
samples from a tropical pine forest.
Figure 3. Rarefaction curves for the three sampling sites.
Figure 4. Dendrogram indicating the relationship among soil
samples according to the identified bacterial strains from nine
clone libraries. Soil sites were compared using Biodiversity Pro
software. The tree was constructed using the Jaccard distance
equation and single linkage method. VO: organic layer of valley;
VT: topsoil of valley; VS: subsoil of valley; MO: organic layer
of middle-slope; MT: topsoil of middle-slope; MS: subsoil of
middle-slope; RO: organic layer of ridge; RT: topsoil of ridge;
and RS: subsoil of ridge.
pg_0009
TSAI et al. ¡X Bacterial community in Fushan Forest soils
65
The higher total species number and diversity of the
ridge samples relative to the valley and middle-slope
samples (Table 3) might be due to the dense vegetation
and floral diversity in this area, which creates specific
niches for a diverse bacterial community. The bacterial
community in the valley was different from that in the
other two sites likely because of the high soil pH and low
soil TOC, TN, and moisture content. Noguez et al. (2005)
also reported that bacterial diversity was high in the hilltop
and middle-slope of two tropical deciduous forests on the
western coast of Mexico. Ng et al. (2006) reported that the
percentages of Proteobacteria and Cyanobacteria in soils
from the Taroko National Park of Taiwan were 36% and
31%, respectively. Further, Cyanobacteria percentages at
high altitudes exceeded those at lower altitudes. The same
phenomenon was also observed in Fushan forest soils,
with the Cyanobacteria only detected in the high altitude
ridge samples.
The bacterial species number was the highest in the
organic layer and decreased through the topsoil and
subsoil. Aeration and organic substrate supply decreased
with the increasing soil depth, leading to reduction of the
bacterial species number and the elimination of bacteria
unable to withstand harsher conditions. The reduction of
bacterial species number in the topsoil and subsoil was
greater in the ridge site than in the valley or middle-slope
site. Other studies using phospholipid fatty acid analysis
(Blume et al., 2002), fluorescence in situ hybridization
(Kobabe et al., 2004), and terminal restriction fragment
length polymorphism analysis (LaMontagne et al., 2003)
have also shown a significant reduction in the species
number of soil microbial communities with changes
in soil depth. Proteobacterial abundance decreased
with the increasing soil depth while Acidobacteria and
Actinobacteria increased with the increasing soil depth.
Surface soils are rich in organic matter from the input of
root exudates, surface litter, and root detritus. This energy
source changes its composition and structure throughout
the profile of a forest soil, from the surface to the deeper
horizons (Agnelli et al., 2002). The rates of C input to
the lower horizons are generally low, and the C tends to
be of limited lability (Trumbore, 2000). In the same way,
the water: air ratio, and the amount of oxygen available
are also limiting factors. Furthermore, the surface soil has
wider variations (both daily and seasonally) in temperature
and moisture than soils at deeper layers (Brady and
Weil, 2002). Such different conditions or gradients in
resource availability and environmental stress of the
pedogenetic horizons could have segregated the microbial
communities. These factors are likely to be the primary
factors for the vertical distribution of the different groups
of bacteria such as Proteobacteria, Acidobacteria, and
Actinobacteria.
The vertical distributions of Gram-negative and Gram-
Table 3. Phylogenetic affiliation of bacterial 16S rDNA clones obtained from the organic layer (O), topsoil (T), and subsoil (S) of
Fushan forest soil at three sampling sites.
Number of 16S rDNA clones
Phylogenetic group
Valley
Middle-slope
Ridge
Total
O T S
O T S
O T S
Proteobacteria, total
22 23 17
23 20 17
24 14 12 172
£\-Proteobacteria
8 7 6
3 10 6
9 2 6 58
£]-Proteobacteria
7 7 5
7 1 2
7 1 2 39
£^-Proteobacteria
2 5 3
8 9 9
7 7 3 53
£_-Proteobacteria
5 4 3
3 2 0
2 2 1 22
Acidobacteria
11 13 18
8 13 11
6 14 19 113
Actinobacteria
2 1 2
3 1 3
2 3 5 22
Bacteroidetes
1 2 0
0 3 3
1 3 1 14
Cyanobacteria
0 0 0
0 0 0
1 0 0
1
Firmicutes
2 1 0
4 3 1
3 2 2 18
Gemmatimonadetes
1 0 0
0 0 0
0 0 0
1
Nitrospirae
1 0 0
0 0 0
0 0 0
1
Planctomycetes
0 0 0
1 0 0
1 0 0
2
TM7 (candidate division)
0 0 0
0 0 3
0 0 0
3
Verrucomicrobia
0 0 0
1 0 0
1 0 0
2
Unclassified, total
0 0 0
0 0 0
1 0 0
1
Total clones
40 40 37
40 40 38
40 36 39 350
Total species number
27 23 18
25 21 19
32 24 19
pg_0010
66
Botanical Studies, Vol. 50, 2009
positive bacteria at different soil layers are consistent with
the patterns observed for other soil profiles: generally,
Gram-negative dominance at the soil surface shifts to
Gram-positive dominance at the deeper soil depths (Blume
et al., 2002; Fierer et al., 2003). These results indicate that
the bacterial communities in the deeper horizons were not
simply diluted analogs of communities in the surface soils
and that some microbes dominated only in the deeper soil
horizons.
Torsvik et al. (1996) demonstrated that information
about the bacterial communities and their diversities are
needed to address the impact of environmental factors on
ecosystem function. Microbial indicators are valuable to
the assessment of soil quality and ecological management
of the forest (Staddon et al., 1999). Future investigation
might address the relationship between soil bacterial
communities and forest ecosystem functions using the
long-term research sites across climatic gradients in the
subtropical zone in Asia.
CONClUSIONS
In summary, the composition of bacterial communities
in Fushan forest soils using a 16S rDNA clone library
was documented, which could be used to construct
special DNA primers and probes to target bacterial
groups of interest. The clone library from Fushan forest
soils represented eleven known bacterial divisions,
Proteobacteria being the most dominant. The ridge soils
exhibited the greatest bacterial species number, suggesting
that the species number is affected by topography.
However, the influence of the floral density and diversity,
which creates specific niches for a variety of organisms,
cannot be underestimated. Further, the increasing
pH through the deeper layers also tends to influence
the existence of different bacteria in different layers.
Bacterial species number was the greatest in the organic
layer and decreased through the deeper soil layers due
to vertical gradient of nutrients, especially the form and
the availability of organic substrates. Differences in the
chemical composition of litter and root exudates might be
expected to affect the availability of carbon sources, which
would subsequently influence the prevalence of microbes.
Acknowledgements. The authors thank Professor Kuo-
Chan Lin, Dr. I-Chu Chen, Mr. Cheng-Hsiung Chang,
and Miss Chia-Bei Wei for their helpful assistance
and the National Science Council of Taiwan for its
financial support (NSC 92-2621-B002-007, NSC
93-2621-B002-005 and NSC 94-2313-B002-090).
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