Accepted Manuscript Title: Characterization of rhizosphere and endophytic bacterial communities from leaves, stems and roots of medicinal Stellera chamaejasme L Author: Hui Jin Xiao-Yan Yang Zhi-Qiang Yan Quan Liu Xiu-Zhuang Li Ji-Xiang Chen Deng-Hong Zhang Li-Ming Zeng Bo Qin PII: DOI: Reference:
S0723-2020(14)00074-5 http://dx.doi.org/doi:10.1016/j.syapm.2014.05.001 SYAPM 25620
To appear in: Received date: Revised date: Accepted date:
26-1-2014 30-4-2014 2-5-2014
Please cite this article as: H. Jin, X.-Y. Yang, Z.-Q. Yan, Q. Liu, X.Z. Li, J.-X. Chen, D.-H. Zhang, L.-M. Zeng, B. Qin, Characterization of rhizosphere and endophytic bacterial communities from leaves, stems and roots of medicinal Stellera chamaejasme L, Systematic and Applied Microbiology (2014), http://dx.doi.org/10.1016/j.syapm.2014.05.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Characterization of rhizosphere and endophytic bacterial
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communities from leaves, stems and roots of medicinal Stellera
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chamaejasme L.
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Hui Jina, Xiao-Yan Yanga, Zhi-Qiang Yana, Quan Liua, Xiu-Zhuang Lia, Ji-Xiang
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Chenb, Deng-Hong Zhangc, Li-Ming Zenga, Bo Qina, d, *
Key Laboratory of Chemistry of Northwestern Plant Resources and Key Laboratory for Natural Medicine of
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a
Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000, China c
School of Petrochemical Engineering, Lanzhou University of Technology, Lanzhou 730050, China
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b
Sino-U.S. Centers for Grazingland Ecosystem Sustainability, Key Laboratory of Grassland Ecosystem Education
Ministry, College of Prataculture, Gansu Agricultural University, Lanzhou 730070, China
State Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou 730000, China
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* Corresponding author at: Key Laboratory of Chemistry of Northwestern Plant Resources and Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, E-mail address:
[email protected] (B. Qin).
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18 Tianshui Middle Road, Lanzhou 730000, China. Tel.: +86 931 4968372; fax: +86 931 8277088.
d
Running title: Rhizosphere and endophytic bacterial communities of S. chamaejasme
CA, correspondence analysis.
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Abbreviations: NA, nutrient agar[CJR1]; OTUs, operational taxonomic units; PCA, principal component analysis;
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ABSTRACT
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A diverse array of bacteria that inhabit the rhizosphere and different plant organs play
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a crucial role in plant health and growth. Therefore, a general understanding of these
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bacterial communities and their diversity is necessary. Using the 16S rRNA gene
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clone library technique, the bacterial community structure and diversity of the
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rhizosphere and endophytic bacteria in Stellera chamaejasme compartments were
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compared and clarified for the first time. Grouping of the sequences obtained showed
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that members of the Proteobacteria (43.2%), Firmicutes (36.5%) and Actinobacteria
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(14.1%) were dominant in both samples. Other groups that were consistently found,
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albeit at lower abundance, were Bacteroidetes (2.1%), Chloroflexi (1.9%), and
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Cyanobacteria (1.7%). The habitats (rhizosphere vs endophytes) and organs (leaf,
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stem and root) structured the community, since the Wilcoxon signed rank test
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indicated that more varied bacteria inhabited the rhizosphere compared to the organs
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of the plant. In addition, correspondence analysis also showed that differences were
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apparent in the bacterial communities associated with these distinct habitats.
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Moreover, principal component analysis revealed that the profiles obtained from the
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rhizosphere and roots were similar, whereas leaf and stem samples clustered together
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on the opposite side of the plot from the rhizosphere and roots. Taken together, these
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results suggested that, although the communities associated with the rhizosphere and organs shared some bacterial species, the associated communities differed in structure and diversity.
Keywords:
Plant-associated bacteria
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Bacterial community structure
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Stellera chamaejasme L.
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16S rRNA gene
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Biodiversity
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Introduction Plants harbor a diverse array of bacteria that inhabit different plant organs and
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tissues, including the rhizosphere, leaves, stems, roots, seeds and fruits [38]. The
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bacteria in the rhizosphere benefit plant growth by increasing the availability of
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mineral nutrients, production of phytohormones, degradation of phytotoxic
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compounds and suppression of pathogens [29,41]. Endophytic bacteria are able to
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contribute to plant growth and development directly or indirectly through biological
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control, plant growth-promoting effects, endophytic nitrogen-fixing activity, and other
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actions [4,49]. Consequently, plant-bacterial associations have gained more and more
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research attention in recent years for their potential biotechnological applications
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[9,35]. Thus, it is crucial to understand the community structure and diversity of
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plant-associated bacteria and their relationships in rhizosphere soil and plant organs.
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Most studies on the structure of the rhizobacteria and plant-associated bacterial
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communities in different species have been performed using both culture-dependent
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and
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biodiversity studies of the rhizosphere and endophytic bacterial community are
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somewhat limited. The major problem is that only a minor fraction of the bacterial
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populations can be recovered [34]. Culture-independent methods, based on analysis of
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DNA extracted directly from rhizosphere and plant samples, represents an alternative
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that overcomes these limitations. Moreover, cultivation-independent methods allow a
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[1,13,51].
However,
culture-dependent
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approaches
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culture-independent
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comparatively rapid analysis of a large number of samples and provide reliable information on the community composition of the rhizosphere and endophytic bacteria [5,42]. Although most of the notable studies on rhizosphere and endophytic bacteria have been focused on crops, horticultural plant species and wild grasses [5,6,8,16], it is still important to explore other plant species for their associated organisms, particularly those of important toxic plants that have the advantage of being able to produce medicines and pesticides [22].
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Stellera chamaejasme L. (Thymelaeaceae), a toxic perennial plant, has a wide
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geographical range from southern Russia to northern China and Mongolia, southwards
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as far as the dry regions of the western Himalayas, the Tibetan Plateau and
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southwestern China [58]. It has become an important pharmacological plant resource,
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and has been used as a raw material for developing various kinds of pesticides and
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medicines. Most studies have demonstrated that extracts of S. chamaejasme show
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significant nematicidal, pesticidal, antimicrobial, phytotoxic, anticancer, antihepatitis
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B virus and immunomodulating activities [10,31,55,57]. Previous ecological studies
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on this toxic plant have been focused on population distribution and dynamics, its
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effect on soil nutrient pools and dynamics, a phylogenetic and phylogeographical
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study, reproductive biology, and endophytic fungi [19,45,58,59]. However, detailed
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knowledge concerning the bacterial community associated with S. chamaejasme is
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still limited.
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Therefore, this study addressed two fundamental questions: (1) How does the
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bacterial community structure differ between the rhizosphere and different organs of S.
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chamaejasme? and (2) Do different habitats, such as rhizosphere and different tissues,
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have the potential to influence the structure of bacterial communities? In order to
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address these questions, the bacterial community structure and diversity in the
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rhizosphere, as well as in leaves, stems and roots of S. chamaejasme were analyzed
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using the 16S rRNA gene clone library technique. The results provided insights into
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the relationships between the rhizosphere and endophytic bacteria in different organs
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of the plant.
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Materials and methods
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Sample collection and surface sterilization
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Samples were collected from the Cuiying mountain area (35°56′ N, 104°08′ E) in
the Yuzhong campus of Lanzhou University on June 8, 2012. Some basic properties of the sampling site were given in the reference of Jin et al. [19]. The sampling site was not privately-owned or protected in any way. It was easy to identify the plant species since S. chamaejasme was in bloom at the sampling time, and 10 healthy looking plants were collected using sterile spades and gloves. The spades were sterilized between different individual plants. The distance between single plants was at least 50
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m depending on the distribution within the vegetation cover. All whole plants were
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immediately put in sterile polystyrene bags and brought to the laboratory on ice.
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The roots of S. chamaejasme were shaken vigorously to separate them from loose
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soil, and the remaining soil closely adhering to the roots (up to 2.5 mm around the
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root) was pooled and considered as rhizosphere soil [24]. One gram of rhizosphere
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soil was weighed from each plant and then pooled as a composite rhizosphere sample
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in sterile laboratory conditions. The composite leaf, stem and root samples were
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processed in a similar way. Healthy, symptomless leaves, stems and roots were
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collected from ten individual S. chamaejasme plants. One gram of leaves, stems and
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roots was weighed from each plant and pooled to make one sample, respectively.
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Samples were numbered Rs for rhizosphere, L for leaf, St for stem, and R for root.
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The leaf, stem, and root samples were rinsed under running tap water for 10 min,
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immersed in 70% ethanol with shaking for 3 min, followed by fresh sodium
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hypochlorite solution (2.5% available Cl-) for 5 min and 70% ethanol for 30 s, and
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finally washed three times with sterile water [46]. Aliquots of the final rinsing water
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were spread on nutrient agar (NA)
medium plates and cultured for 3 d at 28
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[CJR2]solid
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for detection of bacterial colonies in order to examine the effect of the surface
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sterilization. The samples that were not contaminated, based on the culture-dependent
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sterility test, were cut into 0.5-1.0 cm pieces and stored at -80
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DNA-based analyses.
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DNA extraction and PCR amplification of the bacterial 16S rRNA gene
for further
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Total community DNA was extracted from 0.5 g of rhizosphere soil using the ZR
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Soil Microbe DNA KitTM (Zymo Research, Orange, CA, USA), following the
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manufacturer’s instructions. For the endophytic fraction, each sample (0.5 g) was
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frozen with liquid nitrogen, and quickly ground into a fine powder in a pre-cooled
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sterilized mortar and pestle. Each sample was added to the bead tubes from the kit
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following the manufacturer’s instructions. DNA preparations were visualized after electrophoresis in 0.8% agarose gels stained with ethidium bromide under UV light. DNA was further cleaned up by ethanol precipitation and then resuspended in 30 µL sterile Milli-Q water.
Bacterial 16S rRNA genes were PCR amplified using bacteria-specific primers
799F (5′ -AAC AGG ATT AGA TAC CCT G- 3′) and 1492R (5′ -GGT TAC CTT GTT ACG ACT T -3′) that can separate bacterial products and plant chloroplast
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products [6,37]. Each 50 µL PCR contained PCR buffer (Promega, Madison, WI), 50
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ng of DNA template, 2.5 mM MgCl2, 200 µM of each dNTP, 0.5 mg mL-1 bovine
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serum albumin (BSA), 15 pmol of each primer, and 2.5 units Taq polymerase. Cycling
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conditions were 94
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30 sec, 72
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product was isolated by separating the PCR products on 2% low melt agarose gel and
for 2 min, followed by 25 cycles of 94
for 1 min, and with a final extension of 72
for 30 sec, 55
for
for 10 min. The bacterial
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excising a band of agarose with a size of approximately 740 bp. DNA was extracted
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from the gel using the QIAquick gel extraction kit (Qiagen). The concentrations of the
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purified DNA were estimated by comparison with an EZ Load DNA mass standard
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(Bio-Rad).
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Construction of the 16S rRNA gene clone library
The purified PCR-amplified 16S rRNA gene fragments were cloned into the
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pGEM-T vector (Promega, Madison, WI, USA) according to the manufacturer’s
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instructions. Escherichia coli JM109 competent cells (Promega) were transformed
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with the ligation products and spread onto Luria-Bertani (LB) agar plates containing
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ampicillin (100 µg mL-1) and X-gal/IPTG on the surface for standard blue and white
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screening. Approximately 120 positive clones per sample were randomly selected and
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cultured for 24 h at 37
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ampicillin. Plasmid DNA extraction was performed using the QIAprep Spin Miniprep
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Kit (Qiagen) according to the manufacturer’s instructions, and screened by the PCR
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program (described above) using pGEM-T vector-specific primers T7 and SP6. The
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isolated DNAs with correct inserts were confirmed on 0.8% agarose gels and purified
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using the DNA Fragment Quick Purification/Recover Kit (Dingguo, Beijing, China)
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following the protocol provided. Purified PCR products were sequenced with an ABI
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3730 automated sequencer (Invitrogen, Shanghai, China).
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in 1.5 mL of LB broth supplemented with 100 µg mL-1
Sequence analyses and phylogenetic tree construction All the 16S rRNA gene sequences obtained in this study were edited manually using
BioEdit, then aligned with ClustalW and grouped together based on sequence similarity. All assembled sequences were checked for possible chimeric artefacts by the UCHIME algorithm [12]. Potential and identified chimeric sequences were excluded from subsequent analyses. The remaining sequences within each library were grouped into operational taxonomic units (OTUs) based on 3% sequence
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divergence using the furthest-neighbor algorithm in MOTHUR software [39]. The
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get.oturep command in Mothur was used to select representative sequences and they
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were used for all subsequent analyses [39,44]. The taxonomic affiliation of each
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representative sequence was determined by sequence similarity searches against the
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EzTaxon-e database (http://eztaxon-e.ezbiocloud.net/) [23]. Sequences with >97%
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similarity were assigned to the same species. To construct phylogenetic trees, the
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sequences with 97% or higher sequence similarities were aligned with the
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corresponding EzTaxon-e database sequences (93 type strains with greater than 97%
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similarity) from representative members of selected families using the ClustalW
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program. Neighbor-joining phylogenetic trees were constructed from dissimilar
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distance and pairwise comparisons with the Jukes-Cantor distance model using
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MEGA version 5.0 [48]. Bootstrapping with 1,000 replicates was used to assign
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confidence levels to the nodes in the trees.
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Statistical analysis
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The MOTHUR software was applied in order to define OTU rarefaction curves.
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Species richness and diversity indices (Chao 1, non-parametric Shannon’s diversity,
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Simpson diversity index, Shannon’s evenness, and Good’s coverage) were also
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determined by the MOTHUR program at the 0.03 level [39]. The Wilcoxon signed
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rank test was used to test for the significance of differences in the composition of
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bacterial communities from the different habitats [20]. A correspondence analysis
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(CA) was carried out in order to explore patterns of variation in the composition of the
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bacterial community across samples [14]. Principal component analysis (PCA) was
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conducted for evaluating the relationship between bacterial community structures.
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The matrix used for the PCA was a quantitative abundance matrix of all OTUs
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detected from each clone library [25]. The statistical significance for PCA was
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assessed by analysis of variance of PCA scores between different habitats performed
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using SPSS software (version 16.01; SPSS). Nucleotide sequence accession numbers The representative 16S rRNA gene sequences generated by this study were
submitted to GenBank under the accession numbers KF385119-KF385192 (rhizosphere samples), KF385038-KF385088 (leaf samples), KF385193-KF385237 (stem samples) and KF385089-KF385118 (root samples) (Table S1).
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Results and discussion
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Analysis of sequencing data
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The UCHIME algorithm was used to identify sequences with a high probability
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of being chimeric and, as a result, 12 chimeric sequences were discarded. A total of
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468 sequences were obtained (115 from the rhizosphere library, 116 from the leaf
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library, 118 from the stem library and 119 from the root library). Sequences were
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clustered into OTUs based on 3% sequence divergence, which is widely used to
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approximate species-level similarity. It was suggested that the plant-associated
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bacterial community was reasonably well characterized by the sampling effort, since
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the slope of a rarefaction curve was high in the beginning and then gradually
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decreased, causing the curve to level off (Fig. 1). Interestingly, the rarefaction curves
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of the leaf and stem samples were higher than the root samples, while the rhizosphere
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sample was higher than the leaf and stem samples.
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Phylogenetic profiles and taxonomic distribution of the 16S rRNA gene clones among
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the samples
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There were 200 OTUs for all 468 sequences, which were comprised of 74, 51, 45
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and 30 OTUs for the rhizosphere, leaf, stem and root samples, respectively. More than
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80% of the OTUs (164) were composed of a single sequence. However, there were
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five OTUs which contained 20 or more sequences, with OTUl04, OTUst31, OTUr04,
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OTUrs20 and OTUr08 containing 56, 47, 34, 26 and 20 sequences, respectively,
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indicating unevenness of OTU distribution in the bacterial communities (Tables 1 and
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S1). All the 200 representative sequences clustered into eight groups (phyla or classes)
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according to the taxonomic classification of the EzTaxon-e database (Table S1). These
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bacterial
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Proteobacteria
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(43.2%,
including
17.9%
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gamma-subdivision, 16.7% for the alpha-subdivision, 7.5% for the beta-subdivision, and 1.1% for the delta-subdivision), Firmicutes (36.5%), Actinobacteria (14.1%), Bacteroidetes (2.1%), Chloroflexi (1.9%), Cyanobacteria (1.7%), Acidobacteria (0.2%), and Verrucomicrobia (0.2%). These results generally demonstrated high bacterial diversity across the rhizosphere, leaves, stems and roots of S. chamaejasme. The bacterial taxa observed were similar to the findings from other studies that used culture-independent techniques to describe taxa abundances [5,11,50,52].
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Among all representative sequences, most of them showed 99-100% sequence
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similarity, whereas a small number of them showed >97 to 99% sequence similarity.
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More than 97% sequence similarity is accepted for bacterial identification, and