RESEARCH ARTICLE

Vegetation cover of forest, shrub and pasture strongly inuences soil bacterial community structure as revealed by16S rRNA gene T-RFLP analysis
On Chim Chan1, Peter Casper2, Li Qing Sha1, Zhi Li Feng1, Yun Fu1, Xiao Dong Yang1, Andreas Ulrich3 & Xiao Ming Zou1,4
1

Department of Forest Ecosystem, Soil Ecology Group, The Chinese Academy of Sciences, Xishuangbanna Tropical Botanical Garden, Kunming, Yunnan China; 2Department of Limnology of Stratied Lakes, Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Stechlin, Germany; 3Leibniz-Center for Agricultural Landscape Research, Institute of Landscape Matter Dynamics, Muncheberg, Germany; and 4Institute for Tropical Ecosystem Studies, University of Puerto Rico, San Juan, PR, USA

Correspondence: On Chim Chan, Department of Forest Ecosystem, Soil Ecology Group, The Chinese Academy of Sciences, Xishuangbanna Tropical Botanical Garden, 88 Xuefu Road, Kunming, Yunnan 650223, China. Tel.: 186 871 5112637; fax: 186 871 5160916; e-mail: onchim@xtbg.ac.cn Received 15 May 2007; revised 25 February 2008; accepted 26 February 2008. First published online 21 April 2008. DOI:10.1111/j.1574-6941.2008.00488.x Editor: Karl Ritz Keywords bacterial community structure; vegetation type; climate gradient; soil chemical property; T-RFLP.

Abstract Bacterial community structure is inuenced by vegetation, climate and soil chemical properties. To evaluate these inuences, terminal restriction fragment length polymorphism (T-RFLP) and cloning of the 16S rRNA gene were used to analyze the soil bacterial communities in different ecosystems in southwestern China. We compared (1) broad-leaved forest, shrub and pastures in a high-plateau region, (2) three broad-leaved forests representing a climate gradient from highplateau temperate to subtropical and tropical regions and (3) the humus and mineral soil layers of forests, shrub lands and pastures with open and restricted grazing activities, having varied soil carbon and nutrient contents. Principal component analysis of the T-RFLP patterns revealed that soil bacterial communities of the three vegetation types were distinct. The broad-leaved forests in different climates clustered together, and relatively minor differences were observed between the soil layers or the grazing regimes. Acidobacteria dominated the broad-leaved forests (comprising 62% of the total clone sequences), but exhibited lower relative abundances in the soils of shrub (31%) and pasture (23%). Betaproteobacteria was another dominant taxa of shrub land (31%), whereas Alpha- (19%) and Gammaproteobacteria (13%) and Bacteriodetes (16%) were major components of pasture. Vegetation exerted more pronounced inuences than climate and soil chemical properties.

Introduction
The structure of soil bacterial communities is inuenced by vegetation type, climate and soil properties. Microbial communities were found to be different between pasture and forest soils (Nusslein & Tiedje, 1999; Stevenson et al., 2004) and among various forests (Hackl et al., 2004). Sowerby et al. (2005) observed variation of soil microbial extracellular enzyme in heath land ecosystems across geographical and climatic gradients from northern to southern Europe. In addition, microbial communities were reported to respond to alterations in air temperature and humidity (Alekhina et al., 2001; Papatheodorou et al., 2004; Zhang et al., 2005). Soil chemistry also inuences microbial comFEMS Microbiol Ecol 64 (2008) 449458

munity structures. The humus layer has higher carbon and nutrient concentrations than the underlying mineral soil, and grazing intensity affects these same factors in pastures. Bacterial communities have been shown to vary among different forest soil layers (Leckie et al., 2004; Grayston & Prescott, 2005; Chan et al., 2006) and among pasture soils of different grazing intensities (Bardgett et al., 2001; Clegg, 2006). However, studies considering these three factors together are lacking, and it remains unknown whether vegetation type, climate or soil chemistry has a predominant effect on soil bacterial community structure. The landscape of Yunnan province in southwestern China is diverse. Its northwestern high plateau is cool and dry, the mountainous middle has a subtropical climate and the
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southern lowlands are tropical (Qiu & Xie, 1998; Zhu, 2006). Forest, shrub and pasture are representative vegetation types in the high-plateau temperate region, while broad-leaved forest is the dominant natural vegetation in the subtropical and tropical regions. Furthermore, in the high plateau, pasture is managed with open or restricted grazing regimes. In the soils of the Yunnan broad-leaved forests, Acidobacteria have been identied previously as the predominant bacterial phylum (Chan et al., 2006). In this study, we evaluated the degree of similarity of bacterial community structures in (1) soils covered with distinct vegetation types of broad-leaved forest, shrub and pasture at the high-plateau temperate region, (2) the broadleaved forests along geographical locations across a climate gradient and (3) humus and mineral soils of shrub and forest, and pasture of different management regimes, which exhibit different soil carbon and nutrient levels. Terminal restriction fragment length polymorphism (T-RFLP) and cloning analyses of the 16S rRNA gene were used. We explored whether soil bacterial community structures in Yunnan correlated more closely to vegetation type, to geographical location with varying climates or to soil carbon and nutrient contents.

are predominant natural vegetation types in the study regions. Castanopsis wattii and Lithocarpus xylocarpus are the main species at Ailaoshan, and Schima wallichii, Castanopsis hystrix and Lithocarpus fohaiensis are dominant at Xishuangbanna (Kunming Institute of Botany, Chinese Academy of Sciences, 2002; Zhu, 2006). The study sites at Ailaoshan and Xishuangbanna are research stations of the Chinese Ecosystem Research Network, which conducts routine monitoring of various terrestrial, aquatic and atmospheric parameters including climate data (http://www.cern. ac.cn/0index/index.asp). The three study sites are all situated within a 300-km radius.

Soil sampling
Sampling was conducted in July 2004 at Xishuangbanna and Ailaoshan, and in May 2005 at Zhongdian. Humus and mineral soil layers were sampled from the broad-leaved forests and the shrub. In the pasture, only mineral soils were sampled due to the absence of a humus layer. Humus layers were collected with wooden frames (20 20 cm), and mineral soils were taken by a core sampler (diameter: 5 cm, depth: 10 cm). Four independent eld replicates c. 5 m apart were collected at each site. The forests and shrubs are distributed on hillsides and sampling points were selected to follow elevation contours. Each sampling point was represented by a composite of three frames or cores. Samples were transported on ice to the laboratory within 1 day. Soil samples were sieved through 2-mm-mesh sieves. Fresh samples were subjected to chemical analyses. Subsamples were stored at 80 1C for nucleic acids analyses. All soil samples were analyzed by T-RFLP. Cloning and sequencing of 16S rRNA genes was performed on pooled samples of the humus and mineral soil layers of shrub land, and the pasture soils with open and restricted grazing activities. Analyses of the soil samples of broad-leaved forests have been described in Chan et al. (2006).

Materials and methods

Study sites
Zhongdian is a high-plateau temperate region (Table 1) in Yunnan province, southwestern China, where forest, shrub and pasture are three representative vegetation types. The broad-leaved forest is dominated by the species Quercus semicarpifolia, in the family of Fagaceae. Dominant species of shrub are Berberis jamesiana, Rhododendron racemosum and Lyonia ovalifolia, and, in the pasture, Poa partensis and Blysmus sinocompressus (Kunming Institute of Botany, Chinese Academy of Sciences, 2002; Liu, 2006). The shrub is distributed on hillsides adjacent to the pastures. Two grazing management regimes are typically conducted on the pastures: one grazes during the whole year (open grazing) and the other grazes only in winter and spring (restricted grazing). Ailaoshan has a subtropical climate, while Xishuangbanna is tropical. Evergreen broad-leaved forests

Chemical analyses
Soil pH, water content, organic carbon content, total N, hydrolyzable N, extractable NH1-N and NO-N, total P, 4 3 extractable PO3-P and cation exchange capacity (CEC) 4

Table 1. Basic geographical information and type of vegetation of the study sites Study site Zhongdian Location 271N, 991E Elevation (m a.s.l.) 3500 Temperature ( 1C) 5.7 Precipitation (mm) 636 Soil type Alsol Climate Temperate Type of vegetation studied Broad-leaved forest Shrub Pasture Broad-leaved forest Broad-leaved forest

Ailaoshan Xishuangbanna

241N, 1011E 211N, 1011E

2500 800

11.3 22

1931 1556

Alsol Ultisol

Subtropical Tropical

Data are presented as annual average values.

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were analyzed according to State Forestry Administration, P.R. China (1999), which have been described in Chan et al. (2006). Each sample was analyzed in duplicate. Signicant differences between individual samples among the soil chemical properties were evaluated using an unassuming equal variances test Tamhanes T2, one-way ANOVA (P 0.05, SPSS version 13.0).

Bacterial community structure analyzed by T-RFLP

The T-RFLP analysis was performed as described previously in Chan et al. (2006). In brief, total nucleic acids were extracted using an UltraClean Soil DNA Isolation Kit (MoBio Laboratories, Solana Beach) following the manufacturers instructions. The bacterial 16S rRNA gene was PCR amplied using the primers Bact8f (5 0 end labelled with FAM uorescent dye) and Bact926r (Liu et al., 1997), and then digested by HhaI (Amersham Biosciences, Freiburg, Germany) for 4 h at 37 1C. Fluorescently labelled terminal restriction fragments (T-RFs) were size-separated by an automatic sequencer ABI 3100 Genetic Analyzer (Applied Biosystems, Darmstadt, Germany) equipped with a POP6 polymer-lled capillary under denaturing conditions. The fragment length of a T-RF was determined by comparison with the internal standard (GeneScanTM-1000 ROXTM Size Standard, Applied Biosystems). The relative abundance of a single T-RF was calculated as the percentage uorescence intensity relative to the total uorescence intensity of peaks exceeding 1000 uorescence units or 4 2% of the largest peak observed. The proles were standardized according to Dunbar et al. (2001). Briey, the sum of peak heights of each prole was calculated as representation of the total DNA quantity, and the height of each peak was proportionally reduced to the lowest DNA quantity among the samples. The relative intensities of T-RFs from individual samples were subjected to principal component analysis (PCA, MULTI VARIATE STATISTIC PACKAGE, MVSP version 3.1, Kovach Computing Services, Pentraeth, UK) to elucidate major patterns (Blackwood et al., 2003). The scores of the first two components were subsequently used to compare differences between the T-RFLP fingerprint patterns with differences between soil chemical characteristics by calculating Spearman correlation coefficients, and significant differences between major T-RFs were evaluated by Tamhanes T2, one-way ANOVA (P 0.05, SPSS version 13.0). The possible phylogenetic affiliation of a T-RF was assigned based on the corresponding clone library of that soil sample.

ing the manufacturers instructions. 16S rRNA gene sequences were determined using an ABI 3300 sequencer (Applied Biosystems). The sequences obtained were aligned and their phylogenetic relationship was analyzed as described in Chan et al. (2006). Phylogenetic trees were constructed by 100-fold bootstrap analysis by neighborjoining algorithm using the PHYLOGENETIC INFERENCE PACKAGE (PHYLIP) version 3.6 (Felsenstein, 2005). The clone sequences generated in this study were deposited in the GenBank database under the accession nos. EU307119EU307206.

Results
Comparison among distinct vegetation types at the high-plateau region
A representative electropherogram derived from mineral soil of the high-plateau Zhongdian region is illustrated in Fig. 1a. T-RFs with fragment lengths of 35, 61, 90, 182, 195, 283, 292 and 354 bases were dominant; each exhibited 4 5% relative intensity and, in total, contributed over 80% relative fragment abundance. Possible afliations of the T-RFs to phylogenetic groups were assigned based on the clone library established from the broad-leaved forests in a previous study (Chan et al., 2006). The subdivisions in Acidobacteria phyla were classied according to Hugenholtz et al. (1998). For shrub samples, two of the predominant T-RFs in the ngerprints of broad-leaved forest soils, 35 and 61 bases, that were probably afliated to Acidobacteria groups 1, 2 and 5, had only o 2% relative intensities (Fig. 1b). The peak with 283 bases was the most dominant, followed by a 182 base T-RF. Compared with the clone sequences retrieved from shrub land soil (Fig. 3), these T-RFs might afliate to Alpha-, Beta- and Gammaproteobacteria or subdivision 1 of Acidobacteria. As at the shrub site, 35- and 61-base T-RFs were not detected as major fragments in the ngerprints derived from pasture soils (Fig. 1c). Distinctly, a 205-base T-RF appeared as one of the major peaks, and T-RFs at 182 and 195 bases were the two most dominant peaks. The PCA score plot of T-RF data revealed that the soil bacterial community structures of the broad-leaved forest, shrub, and pasture were distinctly separated along the rst and the second principal components, which described 79% and 14% of the total variance, respectively (Fig. 2). In total, 47 and 42 clones were obtained and sequenced from the soils of shrub land and pasture, respectively (Fig. 3). Acidobacteria dominated the broad-leaved forest soils (comprising 62% of the total clone sequences, Chan et al., 2006), but exhibited a lower relative abundance in soils of shrub land (31%) and pasture (23%) (Table 2). Betaproteobacteria was another dominant bacterial class among the clones of shrub land (31%). Over half of these sequences
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Cloning and sequencing analyses

The PCR bacterial amplicons were cloned using a TOPO TA Clonings Kit for Sequencing (Invitrogen, Calsbad) followFEMS Microbiol Ecol 64 (2008) 449458

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(a)

(b)

(c)

Fig. 1. Representative bacterial 16S rRNA gene T-RFLP ngerprint electropherograms derived from (a) the mineral soil of the broad-leaved forest, (b) the mineral soil of the shrub and (c) the pasture with open grazing activity at the Zhongdian study site. Identication of the T-RFs in (a) based on the clone library derived from Ailaoshan and Xishuangbanna forest soils (Chan et al., 2006).

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3 Shrub 2

PC 2 (14% of variation)

Broad-leaved forest 1

0 6 4 2 0 2 4 6

Fig. 2. Score plot of PCA of T-RFLP data. and m, humus and mineral soil of the forest at Zhongdian, respectively, and , humus and mineral soil of the shrub at Zhongdian, respectively, and , pasture at Zhongdian with open and restricted grazing activities, respectively, & and , humus and mineral soil of the forest at Ailaoshan, respectively, and , humus and mineral soil of the forest at Xishuangbanna, respectively.

Pasture

3 PC 1 (79% of variation)

belonged to Burkholderia, and 11% formed a cluster with unidentied afliation. For pasture soil, the major bacterial components were Alphaproteobacteria (afliated to Caulobacter, Bradyrhizobium and Hyphomicrobiaceae), Gammaproteobacteria (belonged to Xanthomonadaceae) and Bacteriodetes. Other bacterial taxa detected were Actinobacteria, Deltaproteobacteria, Gemmatimonadetes and Nitrospira, and three of the sequences were unclassied. 16S rRNA gene sequences retrieved from the soils of shrub and pasture formed separate clusters within the phyla Actinobacteria and Bacteriodetes. Remarkable differences among the soil bacterial communities of forests, shrub and pasture were observed from both cloning and T-RFLP analyses.

paratively low relative intensities of 182-, 283- and 292-base T-RFs. The T-RFLP patterns of the humus and mineral soils of the broad-leaved forests clustered separately (Fig. 2). The 35- and 61-base T-RFs were more dominant in the humus than in the mineral soil layer (P 0.5), and the 182-, 195-, 283-, 292- and 354-base T-RFs were in general lower in the humus than in the mineral soil (Fig. 4). But the difference was less pronounced than among the study sites and among the vegetation types (Fig. 2).

Relationship between soil chemical properties and bacterial community structure

The humus layer had two to three times higher organic carbon, nitrogen and phosphorus contents than the mineral soil of all forest and shrub samples (Table 3). The pasture with open grazing activity exhibited less than half of the organic carbon and nutrient contents than the one with restricted grazing. Similarly, the cation exchange capacities of humus layers were over two to six times higher than mineral soils, and that of the pasture with open grazing activity were also less than half of the one with restricted grazing. These variations between the soil layers and between the grazing intensities were higher than those among the three vegetation types. However, the bacterial T-RFLP patterns from the humus and mineral soils of shrub and from the open and restricted grazing pastures were not separated in the PCA, and the differences among the humus and mineral soils of the forests were only minor (Fig. 2).
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Comparison among broad-leaved forests across a climate gradient

T-RFLP ngerprint patterns derived from the broad-leaved forests in the high-plateau temperate Zhongdian, subtropical Ailaoshan and tropical Xishuangbanna regions differed little, compared with the differences among the three distinct vegetation types of forest, shrub and pasture at Zhongdian (Fig. 2). In the PCA score plot, the forest samples of Zhongdian and Ailaoshan clustered together and were separated from that of Xishuangbanna. The dominant TRFs were similar, but the Xishuangbanna soil samples had distinctly higher relative intensities of 35- and 61-base TRFs, compared with Zhongdian and Ailaoshan forest sites (P 0.5, Fig. 4). Xishuangbanna samples also had comFEMS Microbiol Ecol 64 (2008) 449458

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Aquifex pyrophilus (M83548) S26 P39 Actinobacterium clone FI 1M F10 (EF220617) P24 Actinobacteria P25 Rubrobacteridae bacterium Ellin504 (AY960767) S6 S32 Delta proteobacterium clone GASP WC2W1 A09 (EF075146) Deltaproteobacteria P22 P30 P32 Nitrospirae bacterium clone E06 WMSP2 (DQ450806) Nitrospira P36 P42 Unclassified P9 P15 Bacteroidetes bacterium clone Amb 16S 1087 (EF018461) S41 S29 S31 Bacteroidetes bacterium clone ORSFC2 b08 (EF393416) P40 Bacteroidetes bacterium clone GASP MA1S2 E06 (EF662456) Bacteriodetes P7 P1 S45 P33 P45 P5 P13 Gemmatimonadetes bacterium Ellin7146 (AY673312) Gemmatimonadetes P8 P16 P10 P6 Acidobacteriaceae bacterium Ellin5095 (AY234512) S45 S36 S3 S1 S10 Group S4 S24 1 P12 S49 S21 Acidobacteria Acidobacteria bacterium clone Amb 16S 1563 (EF019024) P21 P26 P17 P14 S22 Acidobacteria bacterium clone Amb 16S 644 (EF018283) P11 Acidobacteria bacterium clone GASP 45KB 168 H09 (EU044408) P44 S7 P3 Group 2 P23 S37 S38 Hyphomicrobiaceae bacterium clone Amb 16S 1766 (EF019133) S47 Hyphomicrobiacaea Hyphomicrobiaceae bacterium clone Elev 16S 1206 (EF019849) S25 S30 P37 Alphaproteobacteria P27 P29 Bradyrhizobiaceae Afipia sp. (RD1 AY296747) P19 Bradyrhizobium sp. clone TM19 6 (DQ303346) Bradyrhizobiaceae S12 S48 S50 Caulobacter sp. A1 (AF361188) P34 Caulobacter P47 P31 P46 Alphapr oteobacterium clone SI 1M G05 (EF221492) P20 Beta pr oteobacterium HTCC304 (AY429720) P18 S35 P43 Burkholderia sp. isolate N3P2 (U37344) S23 Betapr oteobacterium clone Amb 16S 1142 (EF018506) Burkholderia S5 S28 Betaproteobacteria S52 S33 S4 Proteobacterium clone SI 2M H01 (EF221589) S2 S42 S9 S8 S34 S46 Xanthomonadaceae bacterium clone M10Ba23 (AY360613) S13 P35 Gammaproteobacteria P38 P41 (Xanthomonadaceae) S27 Xanthomonadaceae bacterium SG 3 (AF548381) P4 P2 P28 0.1

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Table 2. Relative abundance of bacterial phyla in the soils cover by forest, shrub and pasture in southwest China derived from 16S rRNA clone sequences Relative abundances (%) Forest Acidobacteria Group 1 Group 2 Group 3 Group 5 Group 6 Total Proteobacteria Alpha Beta Gamma Delta Total Bacteriodetes Actinobacteria Gemmatimonadetes Planctomycetes Verrucomicrobia Others 21 22 12 3 4 62 7 6 1 10 24 0 0 0 6 4 4 Shrubw 21 7 2 0 0 30 17 31 5 0 53 10 7 0 0 0 0 Pasturez 15 4 2 2 0 23 19 4 13 6 38 16 6 4 0 0 8

followed by shrub land soils and forest soils at Ailaoshan and Xishuangbanna, and the pasture soils exhibited the lowest C/N ratios. If only the three vegetation types at Zhongdian were considered exclusively, signicant correlations could be observed between the C/N ratios and the rst and second principal components of the PCA (PC 1: r = 0.81, P = 0.001; PC 2: r = 0.48, P = 0.03). However, correlations were found neither with the whole soil sample dataset nor among the three broad-leaved forests across the climate gradient.

Discussion
Our results revealed distinct bacterial community structures among broad-leaved forest, shrub and pasture in the highplateau region of Southwest China. Although identication of the peaks of T-RFLP proles is indenite (Egert & Friedrich, 2003), the remarkable differences of soil bacterial communities among the vegetation types reected from the ngerprint patterns were consistent with our retrieved clone sequences. Acidobacteria, in particular subdivisions 2, 3, 5 and 6, were less abundant in the soils of shrub land and pasture compared with the broad-leaved forests, which accounted for the major difference among these three vegetation types. A shift of dominant bacterial assemblage of Fibrobacter, including Acidobacteria-related sequences, to Proteobacteria was reported after a change of vegetation cover from forest to pasture in Hawaii (Nusslein & Tiedje, 1999). Those authors also reported that differences in bacterial genetic community proles between forest and pasture in Hawaii could be accounted for Burkholderiaand RhizobiumAgrobacterium-afliated clone sequences (Beta- and Alphaproteobacteria, respectively) in pasture soil that are common in the rhizosphere of grasses. In the Amazon region, a higher relative abundance of Acidobacteria-afliated sequences was also found in forest soils than in pasture soils (Borneman & Triplett, 1997). In an arable eld study, Acidobacteria was suggested to participate frequently in bacterial community shifts resulting from soil property changes (Ulrich & Becker, 2006). Acidobacteria is a newly recognized bacterial division (Hugenholtz et al., 1998) that has been shown to be genetically and metabolically diverse and environmentally widespread (Barns et al., 1999), but the potential ecological roles of this bacterial division in the environment remain unknown (Joseph et al., 2003). Apparently, Acidobacteria is more abundant in forest than shrub and pasture soils. In contrast to the investigation in Hawaii by Nusslein & Tiedje (1999), Burkholderia and RhizobiumAgrobacterium were rarely found in our studied pasture soil. But Burkholderia contributed to a substantial portion of the clone library of shrub soils. Sequences associated with the symbiotic nitrogen xer Bradyrhizobium, however, could be retrieved from the soils of all three vegetation types in
 c

Data from Chan et al. (2006).

w Relative abundances are based on 47 clone sequences of pooled samples of the humus and mineral soil layers. z Relative abundances are based on 42 clone sequences of pooled samples of the open and restricted grazing sites.

Soils in the high-plateau region at Zhongdian were drier and less acidic than the forest soils at Ailaoshan and Xishuangbanna. Among the three vegetation types at Zhongdian, the soil pH values and the water contents of the Zhongdian broad-leaved forest and the pasture were similar, while shrub soil had a higher pH and a slightly lower water content. These parameters could not explain the soil bacterial community patterns. No signicant correlations were found between the principal components of the PCA and soil chemical parameters (P 4 0.05). The C/N ratios varied comparatively little between the soil layers and between the pasture management regimes. The forest soils at Zhongdian had the highest C/N ratios,

Fig. 3. Phylogenetic relationship of the bacterial 16S rRNA gene partial sequences retrieved in this study. The sequences denoted as S represent the pooled sample of the humus and the mineral soil layers of shrub, and P denotes the pooled sample of the pasture soils with open and restricted grazing activities. Phylogenetic trees were constructed by 100-fold bootstrap analyses by the neighbor-joining algorithm using PHYLIP. Filled and opened circles at the nodes represent bootstrap values 4 80% and 450%, respectively. The tree is rooted with the rRNA sequences of Aquifex pyrophilus (M83548). The scale bar indicates 10% sequence divergence.

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35%

a Zongdian humus b Zhongdian mineral soil Ailaoshan humus b c,d c d d a a a a,b bb b a b c b a b a Ailaoshan mineral soil Xishuangbanna humus Xishuanbanna mineral soi

30%

25% Relative abundance

c d

20%

15%

a,b a c c b c b,c d e b,c c b

10%
a,b a b a a b

5%

b,c c

0% 35 61 90 182 T-RFs (base) 195 283 292 354

Fig. 4. Relative abundance of dominant T-RFs of the bacterial 16S rRNA gene T-RFLP ngerprint patterns derived from the broad-leaved forests at Zhongdian, Ailaoshan and Xishuangbanna study sites. Error bars represent the SDs of four independent replicates. Different letters denote signicant differences in relative abundances of T-RFs among the soil samples (P 0.05).

southwest China. Besides Proteobacteria, Actinobacteria and Bacteriodetes also accounted for the bacterial community structure differences among the vegetation types. The bacterial community structures among soils of broad-leaved forests across a climate gradient of high plateau temperate, subtropical and tropical regions exhibited higher similarity compared with the various vegetation types at Zhongdian. This pattern was observed even with distinctly different climates of the three study sites, and with the shrub and the pasture located adjacently. Similar observations have been reported by Stevenson et al. (2004). Over a wide geographical range in New Zealand, including both North and South Islands c. 1000 km apart, the catabolic respiration responses of the soil microbial community were reported to be similar among different forest types of podocarp-hardwood, beech and pine forests, but distinct from those of the pastures. Their data supported the conclusion that vegetation cover could be more inuential on the microbial community structure in the soil than geographical locations that differ in climate but are similar in vegetation types. The inuence of the quality of soil organic materials associated with distinct vegetation types (as indicated by the C/N ratio) was stronger than their quantity in affecting the structure of soil bacterial community in the high-plateau region of southwest China. The carbon and nutrient levels of the soil layers and the pasture soils of different grazing management regimes varied considerably, but their bacterial community structures differentiated less pronouncedly than among the vegetation types. Quideau et al. (2001) found a direct link between vegetation and soil organic matter
 c

composition using 13C nuclear magnetic resonance characterization of the chemical structures of soil organics in the Sierra Nevada range in United States. Plant litter quality in forest, shrub and pastures differs markedly. Lignin is a main component of the organic carbon of tree wood debris but grasses are composed mostly of cellulose. In general, lignin has a higher C/N ratio than cellulose, consistent with our C/N ratio observed among the three vegetation covers at the Zhongdian high-plateau region. Thus, forest, shrub and pasture vary considerably in their substrate composition, as do their microbial communities involved in litter degradation (Aneja et al., 2004). Roots of trees and grasses are additional major sources of organic matter to the soil, as well as sites of plant and soil microorganism interactions. The root biomass of forests was found to be higher than pastures in Mexico (Jaramillo et al., 2003a, b) and in the Amazon region (Carvalheiro & Nepstad, 1996). Root exudates inuence the soil bacterial community structure (Baudoin et al., 2003), and their quality and quantity may differ between forest and pasture. Furthermore, excretion from livestock could be an organic input acting as a secondary factor discriminating the bacterial community structures in the soils of the forest and the pasture. Although a decrease of soil acidity and water content was reported in the change of vegetation from forest to pasture in Hawaii (Nusslein & Tiedje, 1999), the pH values and water contents among the forest and pasture at Zhongdian did not show signicant differences and did not correlate with the bacterial community structures in this study. A correlation has been found between soil bacterial communities and carbon and nutrient contents of the
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Table 3. Soil chemical characteristics of the three vegetation types at Zhongdian (broad-leaved forest, shrub and pasture), as well as the forests across the climate gradient in southwest China (from Zhongdian temperate high plateau region, sub-tropical Ailaoshan to tropical Xishuangbanna study sites)

Cation exchange capacity (mol/kg)

24.5 2.3 23.3 6.7 19.6 1.1 16.5 1.5 13.6 3.1 13.9 1.4

16.5 1.2 16.8 0.9

C/N ratiow

17.5 2.3 14.8 2.1

forests at Ailaoshan and Xishuangbanna (Chan et al., 2006). However, these parameters did not explain the bacterial community patterns with the forest soil at Zhongdian in this study. The carbon and nitrogen levels of Zhongdian forest soil were between Ailaoshan and Xishuangbanna, but exhibited a higher C/N ratio. Soil pH and water content also did not correlate to the bacterial community patterns. Other environmental factors, like temperature, might be determinant of soil bacterial community across this climate gradient in Southwest China. The soil bacterial community structures in this study correlated closer to the vegetation types of forest, shrub and pasture than to geographical location with varying climates, or to soil carbon and nutrient contents.

46.88 8.19 6.05 1.44 40.16 1.60 14.66 3.63 15.25 1.11 42.42 9.56

18.17 5.58 3.41 0.21 9.00 2.19 0.23 0.23 1.18 0.10 0.82 0.14

29.76 11.49 1.08 0.31 43.41 19.40 4.44 3.72 0.72 0.62 0.88 0.29

Phosphorous contents

Extractable P (mg-P/kg)

2.23 0.31 2.26 0.67 1.53 0.31 1.24 0.44 1.32 0.06 1.65 0.12

0.34 0.08 0.26 0.04

Total P (g-P/kg)

8.14 8.17 o 0.5

10.11 2.02 2.39 0.52

Acknowledgements
The authors thank the Laboratory for Tropical Rain Forest Ecosystem Research and Management, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, for the nancial support of this study. This research was also supported by the Knowledge Innovation Program of Chinese Academy of Sciences (Grant No. KSCX2-SW-123-5-3). We thank Dr D.Q. Yu, Xishuangbanna Tropical Botanical Garden, for kindly sharing the laboratory facilities, Ms M. Degebrodt, Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Germany, for her excellent technical assistance with T-RFLP analysis, Dr D.A. Schaefer for his helpful assistance with the manuscript preparation and the anonymous journal reviewers for their thorough and pertinent comments.
C/N ratio is calculated by dividing the values of organic carbon by total nitrogen. N/A denotes data not available. Values indicated are the averages and standard deviations of four sampling points.
z

Extractable NO -N (mg-N/kg)

0.95 0.39 o 0.5 1.82 1.46 o 0.5 1.26 0.97 2.04 1.45

5.19 2.08 3.36 0.96 37.03 18.16 10.38 4.27 820 104 505 53 18.38 2.99 6.94 0.98

Extractable NH (mg-N/kg)

41

-N

4.99 1.49 2.38 0.44 6.62 0.99 0.90 0.38 1.89 0.56 1.02 0.3

Hydrolysable N (mg-N/kg)

517 96 246 46 491 37 186 42 323 32 622 97

Nitrogen contents

10.19 1.81 3.44 0.85 8.01 0.77 2.63 0.61 4.63 0.46 11.01 2.09

Total N (g-N/kg)

4.49 1.64 1.64 0.28

334 60 157 26

4.00 0.40 1.46 0.97

1.40 0.59 o 0.5

References
Alekhina LK, Dobrovolskaya TG, Pochatkova TN & Zvyagintsev DG (2001) Evaluation of bacterial diversity in soil microcosms at different moisture. Microbiology 70: 731737. Aneja MK, Sharma S, Munch JC & Schloter M (2004) RNA ngerprinting a new method to screen for differences in plant litter degrading microbial communities. J Microbiol Meth 59: 223231. Bardgett RD, Jones AC, Jones DL, Kemmitt SJ, Cook R & Hobbs PJ (2001) Soil microbial community patterns related to the history and intensity of grazing in sub-montane ecosystems. Soil Biol Biochem 33: 16531664. Barns SM, Takala SL & Kuske CR (1999) Wide distribution and diversity of members of the bacterial kingdom Acidobacterium in the environment. Appl Environ Microbiol 65: 17311737. Baudoin E, Benizri E & Guckert A (2003) Impact of articial root exudates on the bacterial community structure in bulk soil and maize rhizosphere. Soil Biol Biochem 35: 11831192. Blackwood CB, Marsh T, Kim S-H & Paul EA (2003) Terminal restriction fragment length polymorphism data analysis for quantitative comparison of microbial communities. Appl Environ Microbiol 69: 926932.

252.9 66.9 76.9 14.6 157.1 13.7 44.1 14.4 62.7 14.7 153.5 34.9

303.8 57.0 116.1 14.3

Organic C (g-C/kg)

76.8 24.2 23.9 2.3

Values are expressed in dry weight of the soil sample.

Water content (%)

47.2 5.8 38.8 1.8 33.9 5.2 25.7 4.1 45.0 2.8 33.4 4.2

71.9 3.8 57.7 3.5 4.50 0.10 4.22 0.11

5.39 0.12 4.94 0.08 6.54 0.16 5.92 0.75 5.27 0.10 4.95 0.27

pH

Zhongdian Forest, humus Forest, mineral soil Shrub, humus Shrub, mineral soil Pasture, open-grazing Pasture, restricted-grazing Ailaoshan Forest, humus Forest, mineral soil Xishuangbanna Forest, humus Forest, mineral soil

N/Az 4.50 0.10

N/Az 50.0 3.5

FEMS Microbiol Ecol 64 (2008) 449458

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2008 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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FEMS Microbiol Ecol 64 (2008) 449458