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Genetic diversity of endophytic bacteria of the manganese-hyperaccumulating plant Phytolacca americana growing at a manganese mine
European Journal of Soil Biology 62 (2014) 15e21
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European Journal of Soil Biology journal homepage: http://www.elsevier.com/locate/ejsobi
Original article
Genetic diversity of endophytic bacteria of the manganesehyperaccumulating plant Phytolacca americana growing at a manganese mine Yuan Wei, Hong Hou*, YuXian ShangGuan, JiNing Li, FaSheng Li State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese Research Academy of Environmental Science, Beijing 100012, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 June 2013 Received in revised form 22 January 2014 Accepted 17 February 2014 Available online 13 March 2014 Handling editor: Kristina Lindström
Endophytic bacteria have great potential for assisting metal-hyperaccumulating plants in remediation of contaminated soils. However, little information is available on the composition of the endophytic bacterial community of the manganese (Mn)-hyperaccumulator Phytolacca americana. In this study, PCRdenaturing gradient gel electrophoresis was used to analyze the endophytic bacterial diversity and community composition in P. americana growing at an Mn mining site. Results showed that Mn had a significant impact on the bacterial diversity and community structure. Phylogenetic analyses of the recovered DNA sequences classified the bacteria into 10 different divisions, indicating a high level of diversity amongst the endophytic bacterial species of P. americana. Sequencing results demonstrated that Proteobacteria, specifically the g, d and a subclasses, may be the dominant endophytic bacterial genera of P. americana. Some unique sequence types that occurred exclusively in heavily polluted sites were worth investigating their effects on phytoremediation under Mn-contaminated soils. Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Bacterial endophytes Community structure Heavy metal pollution Phytoremediation Phytolacca americana
1. Introduction Contamination of soil with heavy metals has become a worldwide environmental problem, posing a significant threat to human health and ecological security. Phytoremediation, which is the use of plants to extract pollutants from soils, is regarded as an effective, nonintrusive, inexpensive, and socially accepted technology to remediate polluted soils [3,5]. Despite these advantages, many metal-hyperaccumulating plants grow slowly and are inhibited by high concentrations of heavy metals [38,52]. To solve these problems, plant-associated microbes have attracted much attention for their close relationships with host plants. Many studies have demonstrated that they can accelerate seedling emergence and promote plant establishment under adverse conditions, as well as enhance plant growth and development [8,49,54]. Combining hyperaccumulators with plant-associated microbes has more advantages than independent use of hyperaccumulators, and was proposed as one of the most promising green remediation techniques [1,7,44].
* Corresponding author. Tel.: þ86 10 84912638. E-mail addresses: [email protected] (Y. Wei), [email protected] (H. Hou). http://dx.doi.org/10.1016/j.ejsobi.2014.02.011 1164-5563/Ó 2014 Elsevier Masson SAS. All rights reserved.
Endophytic bacteria are plant-associated microbes that ubiquitously inhabit most plant species and do not harm the host plants [41]. Furthermore, recent research demonstrated that they may play an important role in enhancing the tolerance of plants to heavy metals, and increase heavy metal translocation factors, biomass, and trace element concentrations of hyperaccumulators [9,16,27,53]. Thus, endophytic bacteria have great potential for assisting their host plants in remediation of contaminated soils and water [27,50]. Screening for strains of bacteria resistant to heavy metals is a key factor for future application. Endophytic bacteria are highly diverse [45], and plants may be able to select those species that benefit from growth under specific environmental conditions [15,43]. Meanwhile, endophytic bacteria are influenced by the physicochemical properties of the soil and have evolved with the progress of heavy metal contamination [7,36]. Thus, studies of the abundance and composition of bacterial endophytes in the field are essential not only for understanding their interactions with the environment, but also for exploring the possible uses of these bacterial species for the bioremediation of heavy metals. Exposure of animals to excess manganese (Mn) causes Mn toxicity, including Parkinson-like symptoms and abnormalities of the reproductive and immune systems [13,34,48]. Excess Mn in soil can also harm forest and agriculture ecosystems [11,21]. Phytolacca americana is a recently discovered Mn-hyperaccumulating plant
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Table 1 Soil physicochemical properties from the three Mn mine spoils and one adjacent reference area. Samples
TMn (mg/kg)
M1 M2 M3 M4
501 38,838 76,897 88,030
9a 1097 b 3269 c 1266 d
EMn (mg/kg) 75 7165 10,672 11,437
6.6 a 580 b 1998 c 1220 c
pH 7.41 7.23 7.52 7.44
SOM (g/kg)
0.21 0.18 0.23 0.09
a a a a
50.70 48.71 55.16 54.52
4.59 2.93 4.59 2.07
TP (%) a a a a
0.14 0.12 0.14 0.14
EP (mg/kg) 0.01 0.01 0.02 0.01
a a a a
8.42 8.57 8.80 8.50
0.58 0.84 1.85 0.95
TN (%) a a a a
0.24 0.22 0.11 0.06
EN (mg/kg) 0.01 0.01 0.01 0.01
a b c d
122.8 116.8 66.2 33.9
6.0 7.7 5.4 8.5
a a b c
Abbreviations: TMn, total Mn concentration; EMn, extractable Mn concentration; SOM, soil organic matter; TP, total P; EP, extractable P; TN, total N; EN, extractable N. Only the means in the same column are compared. The values marked by different letters were significantly different at a 5% confidence level. Data are means S.E.M.
that has great potential for remediation of Mn-contaminated soils [12,31,35]. To our knowledge, there is little information on the composition of the endophytic bacterial community of P. americana growing in mines or soils highly polluted by Mn. Analysis of the endophytic bacterial diversity is conductive to the biotechnological application of P. americana in combination with endophytic bacteria in Mn-contaminated soil. Molecular techniques, such as polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGGE), and the combination of fingerprints with cloning/ sequencing are proven to be more effective and less biased for the analysis of endophytic bacterial community than cultivationdependent methods [37,40]. The aim of this study was to investigate the diversity of the endophytic bacterial populations in the tissues of P. americana growing in Mn mine by PCR-DGGE. The results of this study provide valuable information on the unique endophytic bacterial species of P. americana, and should aid in development of this hyperaccumulator for remediation of Mncontaminated soils. 2. Materials and methods
with liquid nitrogen and ground to a fine powder in a sterilized and precooled mortar. Total DNA was extracted using a DNA Extraction kit following the manufacturer’s protocol (Axygen Biosciences, China). DNA was dissolved in 100 mL of elution buffer. 2.3. PCR amplification of 16S rRNA genes and denaturing gradient gel electrophoresis (DGGE) analysis A nested PCR was used to investigate the structure of the total endophytic bacterial community. To avoid interference of plant chloroplast DNA, amplification of the 16S rRNA gene was carried out using primers F27 and R1530 in the first round of PCR [23]. Each 25 mL PCR mixture contained 1 mL template solution, 10 mM Trise HCl (pH 8.3), 9.5 mL ddH2O, 10 pmol of each primer (1 mL), and 12.5 mL 2 Master mix (Promega, USA). PCR reactions were performed in a PTC220 gradient DNA thermal cycler (Bio-Rad Laboratories, Hercules, CA, USA) using the following procedure: 94 C for 5 min, followed by 43 cycles of 94 C for 30 s, 52 C for 30 s, 72 C for 90 s, and a final extension at 72 C for 8 min. The PCR products were checked by agarose gel electrophoresis (1.0% (w/v) agarose,
2.1. Sampling of plants and soil Sampling was carried out in the Xiang Tan Mn mining area situated in south central Hunan Province, China (27 530 e28 030 N, 112 450 e112 550 E), where mining and smelting operation ceased in 1913. Based on the distribution of the slag heaps, three Mn mine spoils were selected: a slag heap (site M4), a tailing dam with smelting wastes and wastewater (site M3), and an ore charge heap (site M2). One area (M1), 10 km from the mine area, was chosen as an uncontaminated reference site. Soil and plant samples (fruiting stage) were collected in September 2012 from these areas. Three plots of 10 m 10 m were randomly selected on each site. Each plot was then divided into four 5 m 5 m sampling subplots. The mature leaves and corresponding rhizosphere soils from P. americana were sampled from each subplot. The samples from the four subplots were pooled and homogenized to form a composite sample. The soils and plants were immediately transported to the laboratory and stored at 4 C until further analysis. Portions of the soils were air-dried, ground in a ceramic mortar, and then sieved (2 mm mesh) for analysis of metal and physicochemical traits, following the methods described by Ref. [24]. Leaves were immersed in 70% ethanol for 1 min, washed with sodium hypochlorite solution (2.5% available Cl) for 20 min, rinsed with 70% ethanol for 30 s, and washed three times with sterile distilled water. To confirm complete sterilization, aliquots of the sterile distilled water used in the final rinse were spread on tryptic soy agar plates. The plates were examined for bacterial growth following incubation at 28 C for 7 days. Plant samples that were negative for contamination in this test were used for further analysis. 2.2. Extraction of total bacterial DNA from P. americana Surface-disinfected leaves obtained as described above were used for DNA extraction. Approximately 1 g of leaves was frozen
Fig. 1. DGGE pattern of nested PCR-amplified 16S rDNA fragments of endophytic bacteria from the leaves of P. americana from four sampled sites. The labeled bands in the gel correspond to the sequenced clones.
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reamplified with primer pair F341/R534. PCR products were purified with a PCR Cleanup Kit (Axygen Biosciences) and sent to Shanghai Sangon Biological Engineering Technology & Services Company for sequencing (Shanghai, China), where F341 and R534 were used as sequencing primers. 2.5. Data analysis
Fig. 2. ShannoneWeiner diversity index of endophytic bacterial communities generated from DGGE band pattern. The columns marked by the same letter indicate nonsignificant differences at the confidence level of 0.05. Data are means S.E.M.
100 V, 60 min). Amplification products from the first round of PCR were diluted 10-fold and 1 mL of this dilution was used as the template for the second round of PCR using primers F341-GC and R534, with the same reaction ingredients as the first PCR reaction [40]. Thermal cycling consisted of an initial denaturation at 95 C for 5 min, followed by 35 cycles of 95 C for 30 s, 55 C for 45 s, 72 C for 90 s, and a final extension at 72 C for 10 min. The nested PCR amplicons were checked by agarose gel electrophoresis (1.5% (w/v) agarose, 100 V, 60 min) and ethidium bromide staining to determine product size (approximately 200 bp) and yield, with a pBR322 DNA/Alul marker. Twenty microliters of nested PCR products were used for DGGE analysis. The gels contained 8% (w/v) polyacrylamide (37:1 acrylamide/bis-acrylamide) and 1 TriseAcetateeEDTA buffer and were 1.5 mm thick (20 cm 20 cm). A linear gradient from 35% to 55% denaturant was used, where 100% denaturing acrylamide was defined as containing 7 M urea and 40% formamide [32]. All DGGE analysis was run using a D-Gene system (Bio-Rad Laboratories) at a constant temperature of 60 C. Electrophoresis was run for 10 min at 200 V, after which the voltage was lowered to 150 V for an additional 6 h. Gels were stained using AgNO3 staining solution and gel images were digitally captured using a ChemiDoc EQ system (Bio-Rad Laboratories). 2.4. Sequence analysis Prominent DGGE bands were excised from the acrylamide gel and the DNA was eluted using a Poly-Gel DNA Extraction Kit (Omega Scientific, Tarzana, CA, USA). Eluted DNA (1 mL) was
DGGE profiles of amplified endophytic bacterial fragments from different samples were analyzed by Bio-Rad Quantity One 4.4.0 software. The ShannoneWeiner index (H) was used to characterize endophytic bacterial diversity [25], using the formula: P H ¼ Si¼ 1 Pi ln pi, where H ¼ ShannoneWeiner index, S ¼ total number of bands in each sample, and Pi is the relative abundance of the i-th band of each sample. Soil physicochemical property and diversity index data were analyzed by one-way ANOVA following a homogeneity test. Least significant difference (LSD) at the 5% confidence level was performed to compare the mean difference between various sampling sites. Two-tailed Pearson correlation analysis was used to analyze the relationship between various soil properties and endophytic bacterial diversity. The presence or absence of the band in a DGGE profile was used to construct a two-dimensional matrix, which was interpreted by principal component analysis (PCA). All analyses were performed using SPSS v.10.0 software (SPSS Inc., Chicago, IL, USA). Sequences were compared with known sequences using the basic local alignment search tool (BLAST, http://www.ncbi.nlm.nih. gov/BLAST/ [2], and the nearest neighbor bacterial sequences were aligned with sample 16S rRNA sequences using ClustalX 1.83. A phylogenetic tree was inferred by the neighbor-joining method using Mega 4.0 software [46]. The nucleotide accession numbers for the sequences reported in this paper are listed in Table 3. 3. Results 3.1. Metal and physicochemical traits Soil physicochemical properties of the four sampling sites are shown in Table 1. Site M4 possessed the highest Mn concentrations (both total and extractable Mn), and Site M1 (the reference site) had the lowest Mn concentrations (both total and extractable Mn). When compared with the background level of Mn in soils from Hunan Province (Mn ¼ 459 mg/kg) [28], the Mn contents in the three Mn mine spoils were 85-fold (M2), 165-fold (M3), and 191fold (M4) greater, respectively. The Mn content in soil from reference site M1 was similar to the background value.
Table 2 Correlation analysis of ShannoneWeiner index and soil physicochemical properties.
H TMn EMn pH SOM TP EP TN EN
H
TMn
EMn
pH
SOM
TP
EP
TN
EN
1 0.973** 0.911** 0.288 0.568 0.277 0.048 0.993** 0.959**
1 0.976** 0.237 0.507 0.143 0.077 0.951** 0.897**
1 0.095 0.401 0.064 0.002 0.877** 0.811**
1 0.675* 0.056 0.704* 0.317 0.259
1 0.154 0.348 0.588* 0.537
1 0.311 0.344 0.341
1 0.012 0.029
1 0.976**
1
**Denotes significant correlation at p < 0.01. *Denotes significant correlation at p < 0.05. Abbreviations: H, ShannoneWeiner index; TMn, total Mn concentration; EMn, extractable Mn concentration; SOM, soil organic matter; TP, total P; EP, extractable P; TN, total N; EN, extractable N.
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3.2. DGGE analysis
Table 3 GenBank homologies for sequences from recovered DGGE bands.
Nested PCR amplifications were successful, and endophytic bacterial communities associated with different leaf samples were observed on the DGGE gel (Fig. 1). Overall, the band number, intensity, and composition in various sites showed significant differences. Some bands, such as 7, 8, 9, 10, and 17, were detected in all sites. Bands 1 and 5 were present only in site M1. Bands 2, 3, 6, 14, and 15 were not detected in sites M3 and M4, while band 11 was not detected in sites M1 and M2. Site M4 contained some unique bands, such as 12 and 13, that were absent in other sites. Meanwhile, the common bands from site M1 were more intense than from other sites. The ShannoneWeiner index was calculated based on the proportional intensity of bands. Community M1 had the highest diversity index, followed by M2, M3, and then M4 (Fig. 2). The ShannoneWeiner index between various sites was significantly different. Correlation analysis (Table 2) showed that the ShannoneWeiner index was significantly negatively correlated with total soil Mn concentration and extractable Mn concentration (p < 0.01), and significantly positively correlated with total soil nitrogen concentration and extractable nitrogen concentration (p < 0.01). PCA showed that the first two components explained 81.6% of the variance in the endophytic bacterial community. PC1 and PC2 explained 44.1% and 37.4% of the total variance, respectively. Endophytic bacterial communities from four sites were distinctly clustered into four groups, with M1 in the second quadrant, M2 in the third quadrant, M3 in the first quadrant, and M4 in the fourth quadrant (Fig. 3).
Band name
Genbank The nearest BLAST matches accession no.
WY WY WY WY WY WY WY WY WY WY WY WY WY WY
KC410811 KC410814 KC410818 KC410820 KC410823 KC410826 KC410829 KC410832 KC410835 KC410839 KC410841 KC410844 KC410848 KC410850
1-1 2-1 3-3 4-1 5-1 6-1 7-1 8-1 9-1 10-2 11-1 12-1 13-2 14-2
WY 15-2 KC410853 WY 16-1 KC410856 WY 17-2 KC410859 WY 18-1 KC410862
Similarity %
Uncultured Syntrophus sp (JN392959) 98% Sphingopyxis sp (JQ658411) 99% Pseudomonas putida (JX401461) 99% Uncultured delta proteobacterium (HM438022) 98% Uncultured Bacteroidetes bacterium (JX240980) 100% Uncultured Ohtaekwangia sp (JQ771971) 97% Uncultured Actinomyces sp (AY435193) 99% Uncultured Chloroflexi bacterium (JN038250) 99% Filobacillus milosensis (NR027209) 99% Alkalilimnicola ehrlichii (CP000453) 98% Marinobacter lipolyticus (JN202615) 94% Lysobacter spongiicola (NR041587) 95% Uncultured bacterium (HQ190505) 97% Uncultured gamma proteobacterium 98% (JF727680) Uncultured Gemmatimonadetes bacterium 97% (AY795668) Salinisphaera sp (AB735546) 96% Micrococcus yunnanensis (JX885670) 100% Uncultured Acidobacteria bacterium 99% (HM447889)
statistical analysis (Fig. 5) showed that bands 1, 2, 3, 4, 10, 11, 12, 14, and 16 belong to Proteobacteria, bands 5 and 6 belong to Bacteroidetes, bands 7 and 17 belong to Actinobacteria, and all others belong to distinct groups, respectively. The sequencing results and their phylogenetic relationship suggested that endophytic bacterial species in the leaves of P. americana were abundant and diverse.
3.3. Sequence and phylogenetic analysis 4. Discussion Eighteen bands of interest were excised from the DGGE gel for cloning and sequencing (Table 3). A phylogenetic reconstruction of these cloned sequences, together with sequences from their closest relatives from the GenBank database, is shown in Fig. 4. Further
Studies of the composition of endophytes of hyperaccumulating plants living in a contaminated environment have attracted much attention because of their biotechnological applications in
Fig. 3. Principal component analysis of endophytic bacterial communities generated from DGGE band patterns.
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Fig. 4. Neighbor-joining phylogenetic tree of partial 16S rRNA gene sequences amplified from samples of leaves from P. americana. Bootstrap support values with 1000 replicates are given along the branches.
bioremediation [17,29]. Many metal-resistant endophytic bacteria have been isolated from various hyperaccumulators, such as Elsholtzia splendens (copper), Alyssum bertolonii (nickel), and Solanum nigrum L. (cadmium) [4,26,44]. However, little attention has been paid to the composition of endophytic bacterial communities of Mn-hyperaccumulators growing naturally on Mn mine sites. In this study, we explored the diversity of the endophytic bacterial community associated with the Mn-hyperaccumulator P. americana in an Mn mine by a culture-independent approach. The sample sites were highly polluted by Mn (Table 1), However, P. americana grew well and endophytic bacteria were detected in its tissues, which suggested they had adapted to such heavy metal stress through coevolution over an extended period. The endophytic
bacteria found in this experiment are likely to have high Mn tolerance, and might play an important role in improving P. americana growth and health under significant Mn stress. The ShannoneWeiner index decreased significantly with the increasing Mn concentrations from M1 to M4 (Fig. 2). Meanwhile, correlation analysis showed that the ShannoneWeiner index was significantly negatively correlated with total soil Mn concentration and extractable Mn concentration at p < 0.01 (Table 2). Further PCA analysis showed that endophytic bacterial communities from the four sites scattered in four different quadrants, indicating that the community structure of the endophytic bacteria had changed markedly. Heavy metal pollution can change the size, composition, and activity of plant-associated microbial communities [14,52].
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Fig. 5. Distribution of the bacterial category of endophytic bacterial 16S rDNA clone libraries of P. americana.
Some studies have shown that the pollutant was a dominating factor influencing endophytic bacterial community structure [20]. Our results suggested that the diversity of endophytic bacteria in P. americana at Mn mine sites should be impacted by Mn contamination. Nevertheless, it should be noted that the ShannoneWeiner index was also significantly correlated with soil nitrogen concentration, which indicated soil Mn concentration was not the sole factor influencing endophytic bacterial diversity. A plant’s bacterial endophyte community is thought to be recruited from the rhizosphere, so it is also influenced by the physicochemical properties of the soil [36]. The results of our study suggested soil properties, especially nitrogen, also contributed to the variation of endophytic bacterial diversity in P. americana. A more interesting thing is that nitrogen and Mn concentrations are indeed significantly negatively correlated across the sampling sites. Many studies demonstrated heavy metal pollution can influence soil nitrogen contents and chemical forms through inhibiting nitrogen mineralization, nitrification, denitrification and biological nitrogen fixation [33,51]. In this study, nitrogen concentrations decreased with the increasing Mn concentrations which indicated long term Mn pollution may influence soil nitrogen concentration and chemical forms, and further influence endophytic bacterial diversity. In general, the factors influencing endophytic bacterial diversity are varied and their interactions are complex, especially in the field. Therefore, further research is needed to find the exact relationship between environmental factors and endophytic bacteria of P. americana. It is well known that the bacteria isolated from polluted environments are more tolerant to higher concentrations of metals than those isolated from unpolluted areas [18,42]. Further, metal tolerance of bacterial communities increases with the addition of metals, by the death of sensitive species and subsequent competition and adaptation of surviving bacteria [10,39]. Some DGGE bands in this study disappeared with the increasing Mn concentrations from sites M1 to M4, while other bands appeared at the higher Mn concentrations. These unique endophytic bacteria sequence types were intriguing. The sequence types found exclusively in site M1 (such as bands 1 and 5) may represent Mnsensitive endophytic bacterial species. Bands that disappeared in sites M3 and M4 (such as bands 3 and 14) represent species that can only endure relatively light Mn pollution, while the sequence types found exclusively in sites M3 and M4 (such as bands 11, 12, and 13) may be indicative of highly tolerant endophytic bacterial species. As previously mentioned, the influence mechanisms of Mn pollution on these endophytic bacterial species are complex. One possible mechanism is that Mn influence endophytic bacterial species through impacting soil nitrogen concentration and chemical forms
indirectly. Furthermore, band 12 belonged to Lysobacter, which can produce an antibiotic and suppress plant pathogens [19,22]. Band 13 was related to an uncultured bacterial species, implying that it might be a novel genera that is emerging through extensive coevolution with P. americana. Interactions between P. americana and these special endophytes may be important for survival and growth of plants in highly Mn-contaminated soils. The highly tolerant endophytic bacterial species associated with P. americana may have extensive application in improving phytoremediation efficiency in Mn-contaminated soils [6,44]. One question that needs to be explored is only 3 samples from one uncontaminated soil were analyzed which is limited for comparison researching. Some special species may cannot be discovered as this reason. Thus, it is worthwhile to conducted more extensive investigation in future work. Different hyperaccumulators can harbor different endophytic bacterial populations. Chen analyzed the diversity of bacterial endophytes associated with Cd-hyperaccumulator S. nigrum L. growing in mine tailing. Sphingomonas and Pseudomonas were found to be dominant genera of S. nigrum L. [7]. In this study, phylogenetic analysis revealed that the majority of clones were affiliated with Proteobacteria (50%, nine bands), which included g (33.3%, six bands), d (11.1%, two bands), and a (5.6%, one band) subclasses. The sequences of other clones showed they belong to Bacteroidetes (11.1%, two bands), Actinobacteria (11.1%, two bands), Chloroflexi (5.6%, one band), Bacilli (5.6%, one band), Gemmatimonadetes (5.6%, one band), and Acidobacteria (5.6%, one band), respectively. In addition, 5.6% (one band) of the sequences was unclassified, but showed high similarity to uncultured bacterial sequences (Fig. 4). Our results demonstrated that Proteobacteria may be the dominant endophytic bacterial genus of P. americana. Proteobacteria also make up the largest fraction of the clone library of other endophytic bacterial communities [47]. Further, the biological functions of Proteobacteria are varied, including growth promotion, pollutant degradation, and assistance with phytoremediation of heavy metals [30]. 5. Conclusions All of the endophytic bacteria detected in the current study were classified into 10 different divisions, suggesting that endophytic bacterial species of P. americana are abundant and diverse. The phylogenetic analysis revealed that different species occupied remarkably different genetic positions, which might have different implications for their performance under Mn-contaminated conditions. Our future work will be to isolate endophytic bacterial strains from P. americana and cultivate them independently to investigate their effects on phytoremediation under controlled conditions. Acknowledgments This study was supported by grants from the National Natural Science Foundation of China (41271338, 41303066) and the Special Environmental Protection Foundation for Public Welfare Project (201009032) and the National Science Foundation for Post-doctoral Scientists of China (2013M530685) and the State Key Laboratory Program, Chinese Research Academy of Environmental Science (SKLECRA2013OFP03). Special thanks to Professor Zeng (Hunan Agriculture University) for his assistance in the sampling process. References [1] E.R. Alford, E.A.H. Pilon-Smits, M.W. Paschke, Metallophytes: a view from the rhizosphere, Plant Soil 337 (2010) 33e50.
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Contents lists available at ScienceDirect
European Journal of Soil Biology journal homepage: http://www.elsevier.com/locate/ejsobi
Original article
Genetic diversity of endophytic bacteria of the manganesehyperaccumulating plant Phytolacca americana growing at a manganese mine Yuan Wei, Hong Hou*, YuXian ShangGuan, JiNing Li, FaSheng Li State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese Research Academy of Environmental Science, Beijing 100012, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 June 2013 Received in revised form 22 January 2014 Accepted 17 February 2014 Available online 13 March 2014 Handling editor: Kristina Lindström
Endophytic bacteria have great potential for assisting metal-hyperaccumulating plants in remediation of contaminated soils. However, little information is available on the composition of the endophytic bacterial community of the manganese (Mn)-hyperaccumulator Phytolacca americana. In this study, PCRdenaturing gradient gel electrophoresis was used to analyze the endophytic bacterial diversity and community composition in P. americana growing at an Mn mining site. Results showed that Mn had a significant impact on the bacterial diversity and community structure. Phylogenetic analyses of the recovered DNA sequences classified the bacteria into 10 different divisions, indicating a high level of diversity amongst the endophytic bacterial species of P. americana. Sequencing results demonstrated that Proteobacteria, specifically the g, d and a subclasses, may be the dominant endophytic bacterial genera of P. americana. Some unique sequence types that occurred exclusively in heavily polluted sites were worth investigating their effects on phytoremediation under Mn-contaminated soils. Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Bacterial endophytes Community structure Heavy metal pollution Phytoremediation Phytolacca americana
1. Introduction Contamination of soil with heavy metals has become a worldwide environmental problem, posing a significant threat to human health and ecological security. Phytoremediation, which is the use of plants to extract pollutants from soils, is regarded as an effective, nonintrusive, inexpensive, and socially accepted technology to remediate polluted soils [3,5]. Despite these advantages, many metal-hyperaccumulating plants grow slowly and are inhibited by high concentrations of heavy metals [38,52]. To solve these problems, plant-associated microbes have attracted much attention for their close relationships with host plants. Many studies have demonstrated that they can accelerate seedling emergence and promote plant establishment under adverse conditions, as well as enhance plant growth and development [8,49,54]. Combining hyperaccumulators with plant-associated microbes has more advantages than independent use of hyperaccumulators, and was proposed as one of the most promising green remediation techniques [1,7,44].
* Corresponding author. Tel.: þ86 10 84912638. E-mail addresses: [email protected] (Y. Wei), [email protected] (H. Hou). http://dx.doi.org/10.1016/j.ejsobi.2014.02.011 1164-5563/Ó 2014 Elsevier Masson SAS. All rights reserved.
Endophytic bacteria are plant-associated microbes that ubiquitously inhabit most plant species and do not harm the host plants [41]. Furthermore, recent research demonstrated that they may play an important role in enhancing the tolerance of plants to heavy metals, and increase heavy metal translocation factors, biomass, and trace element concentrations of hyperaccumulators [9,16,27,53]. Thus, endophytic bacteria have great potential for assisting their host plants in remediation of contaminated soils and water [27,50]. Screening for strains of bacteria resistant to heavy metals is a key factor for future application. Endophytic bacteria are highly diverse [45], and plants may be able to select those species that benefit from growth under specific environmental conditions [15,43]. Meanwhile, endophytic bacteria are influenced by the physicochemical properties of the soil and have evolved with the progress of heavy metal contamination [7,36]. Thus, studies of the abundance and composition of bacterial endophytes in the field are essential not only for understanding their interactions with the environment, but also for exploring the possible uses of these bacterial species for the bioremediation of heavy metals. Exposure of animals to excess manganese (Mn) causes Mn toxicity, including Parkinson-like symptoms and abnormalities of the reproductive and immune systems [13,34,48]. Excess Mn in soil can also harm forest and agriculture ecosystems [11,21]. Phytolacca americana is a recently discovered Mn-hyperaccumulating plant
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Table 1 Soil physicochemical properties from the three Mn mine spoils and one adjacent reference area. Samples
TMn (mg/kg)
M1 M2 M3 M4
501 38,838 76,897 88,030
9a 1097 b 3269 c 1266 d
EMn (mg/kg) 75 7165 10,672 11,437
6.6 a 580 b 1998 c 1220 c
pH 7.41 7.23 7.52 7.44
SOM (g/kg)
0.21 0.18 0.23 0.09
a a a a
50.70 48.71 55.16 54.52
4.59 2.93 4.59 2.07
TP (%) a a a a
0.14 0.12 0.14 0.14
EP (mg/kg) 0.01 0.01 0.02 0.01
a a a a
8.42 8.57 8.80 8.50
0.58 0.84 1.85 0.95
TN (%) a a a a
0.24 0.22 0.11 0.06
EN (mg/kg) 0.01 0.01 0.01 0.01
a b c d
122.8 116.8 66.2 33.9
6.0 7.7 5.4 8.5
a a b c
Abbreviations: TMn, total Mn concentration; EMn, extractable Mn concentration; SOM, soil organic matter; TP, total P; EP, extractable P; TN, total N; EN, extractable N. Only the means in the same column are compared. The values marked by different letters were significantly different at a 5% confidence level. Data are means S.E.M.
that has great potential for remediation of Mn-contaminated soils [12,31,35]. To our knowledge, there is little information on the composition of the endophytic bacterial community of P. americana growing in mines or soils highly polluted by Mn. Analysis of the endophytic bacterial diversity is conductive to the biotechnological application of P. americana in combination with endophytic bacteria in Mn-contaminated soil. Molecular techniques, such as polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGGE), and the combination of fingerprints with cloning/ sequencing are proven to be more effective and less biased for the analysis of endophytic bacterial community than cultivationdependent methods [37,40]. The aim of this study was to investigate the diversity of the endophytic bacterial populations in the tissues of P. americana growing in Mn mine by PCR-DGGE. The results of this study provide valuable information on the unique endophytic bacterial species of P. americana, and should aid in development of this hyperaccumulator for remediation of Mncontaminated soils. 2. Materials and methods
with liquid nitrogen and ground to a fine powder in a sterilized and precooled mortar. Total DNA was extracted using a DNA Extraction kit following the manufacturer’s protocol (Axygen Biosciences, China). DNA was dissolved in 100 mL of elution buffer. 2.3. PCR amplification of 16S rRNA genes and denaturing gradient gel electrophoresis (DGGE) analysis A nested PCR was used to investigate the structure of the total endophytic bacterial community. To avoid interference of plant chloroplast DNA, amplification of the 16S rRNA gene was carried out using primers F27 and R1530 in the first round of PCR [23]. Each 25 mL PCR mixture contained 1 mL template solution, 10 mM Trise HCl (pH 8.3), 9.5 mL ddH2O, 10 pmol of each primer (1 mL), and 12.5 mL 2 Master mix (Promega, USA). PCR reactions were performed in a PTC220 gradient DNA thermal cycler (Bio-Rad Laboratories, Hercules, CA, USA) using the following procedure: 94 C for 5 min, followed by 43 cycles of 94 C for 30 s, 52 C for 30 s, 72 C for 90 s, and a final extension at 72 C for 8 min. The PCR products were checked by agarose gel electrophoresis (1.0% (w/v) agarose,
2.1. Sampling of plants and soil Sampling was carried out in the Xiang Tan Mn mining area situated in south central Hunan Province, China (27 530 e28 030 N, 112 450 e112 550 E), where mining and smelting operation ceased in 1913. Based on the distribution of the slag heaps, three Mn mine spoils were selected: a slag heap (site M4), a tailing dam with smelting wastes and wastewater (site M3), and an ore charge heap (site M2). One area (M1), 10 km from the mine area, was chosen as an uncontaminated reference site. Soil and plant samples (fruiting stage) were collected in September 2012 from these areas. Three plots of 10 m 10 m were randomly selected on each site. Each plot was then divided into four 5 m 5 m sampling subplots. The mature leaves and corresponding rhizosphere soils from P. americana were sampled from each subplot. The samples from the four subplots were pooled and homogenized to form a composite sample. The soils and plants were immediately transported to the laboratory and stored at 4 C until further analysis. Portions of the soils were air-dried, ground in a ceramic mortar, and then sieved (2 mm mesh) for analysis of metal and physicochemical traits, following the methods described by Ref. [24]. Leaves were immersed in 70% ethanol for 1 min, washed with sodium hypochlorite solution (2.5% available Cl) for 20 min, rinsed with 70% ethanol for 30 s, and washed three times with sterile distilled water. To confirm complete sterilization, aliquots of the sterile distilled water used in the final rinse were spread on tryptic soy agar plates. The plates were examined for bacterial growth following incubation at 28 C for 7 days. Plant samples that were negative for contamination in this test were used for further analysis. 2.2. Extraction of total bacterial DNA from P. americana Surface-disinfected leaves obtained as described above were used for DNA extraction. Approximately 1 g of leaves was frozen
Fig. 1. DGGE pattern of nested PCR-amplified 16S rDNA fragments of endophytic bacteria from the leaves of P. americana from four sampled sites. The labeled bands in the gel correspond to the sequenced clones.
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reamplified with primer pair F341/R534. PCR products were purified with a PCR Cleanup Kit (Axygen Biosciences) and sent to Shanghai Sangon Biological Engineering Technology & Services Company for sequencing (Shanghai, China), where F341 and R534 were used as sequencing primers. 2.5. Data analysis
Fig. 2. ShannoneWeiner diversity index of endophytic bacterial communities generated from DGGE band pattern. The columns marked by the same letter indicate nonsignificant differences at the confidence level of 0.05. Data are means S.E.M.
100 V, 60 min). Amplification products from the first round of PCR were diluted 10-fold and 1 mL of this dilution was used as the template for the second round of PCR using primers F341-GC and R534, with the same reaction ingredients as the first PCR reaction [40]. Thermal cycling consisted of an initial denaturation at 95 C for 5 min, followed by 35 cycles of 95 C for 30 s, 55 C for 45 s, 72 C for 90 s, and a final extension at 72 C for 10 min. The nested PCR amplicons were checked by agarose gel electrophoresis (1.5% (w/v) agarose, 100 V, 60 min) and ethidium bromide staining to determine product size (approximately 200 bp) and yield, with a pBR322 DNA/Alul marker. Twenty microliters of nested PCR products were used for DGGE analysis. The gels contained 8% (w/v) polyacrylamide (37:1 acrylamide/bis-acrylamide) and 1 TriseAcetateeEDTA buffer and were 1.5 mm thick (20 cm 20 cm). A linear gradient from 35% to 55% denaturant was used, where 100% denaturing acrylamide was defined as containing 7 M urea and 40% formamide [32]. All DGGE analysis was run using a D-Gene system (Bio-Rad Laboratories) at a constant temperature of 60 C. Electrophoresis was run for 10 min at 200 V, after which the voltage was lowered to 150 V for an additional 6 h. Gels were stained using AgNO3 staining solution and gel images were digitally captured using a ChemiDoc EQ system (Bio-Rad Laboratories). 2.4. Sequence analysis Prominent DGGE bands were excised from the acrylamide gel and the DNA was eluted using a Poly-Gel DNA Extraction Kit (Omega Scientific, Tarzana, CA, USA). Eluted DNA (1 mL) was
DGGE profiles of amplified endophytic bacterial fragments from different samples were analyzed by Bio-Rad Quantity One 4.4.0 software. The ShannoneWeiner index (H) was used to characterize endophytic bacterial diversity [25], using the formula: P H ¼ Si¼ 1 Pi ln pi, where H ¼ ShannoneWeiner index, S ¼ total number of bands in each sample, and Pi is the relative abundance of the i-th band of each sample. Soil physicochemical property and diversity index data were analyzed by one-way ANOVA following a homogeneity test. Least significant difference (LSD) at the 5% confidence level was performed to compare the mean difference between various sampling sites. Two-tailed Pearson correlation analysis was used to analyze the relationship between various soil properties and endophytic bacterial diversity. The presence or absence of the band in a DGGE profile was used to construct a two-dimensional matrix, which was interpreted by principal component analysis (PCA). All analyses were performed using SPSS v.10.0 software (SPSS Inc., Chicago, IL, USA). Sequences were compared with known sequences using the basic local alignment search tool (BLAST, http://www.ncbi.nlm.nih. gov/BLAST/ [2], and the nearest neighbor bacterial sequences were aligned with sample 16S rRNA sequences using ClustalX 1.83. A phylogenetic tree was inferred by the neighbor-joining method using Mega 4.0 software [46]. The nucleotide accession numbers for the sequences reported in this paper are listed in Table 3. 3. Results 3.1. Metal and physicochemical traits Soil physicochemical properties of the four sampling sites are shown in Table 1. Site M4 possessed the highest Mn concentrations (both total and extractable Mn), and Site M1 (the reference site) had the lowest Mn concentrations (both total and extractable Mn). When compared with the background level of Mn in soils from Hunan Province (Mn ¼ 459 mg/kg) [28], the Mn contents in the three Mn mine spoils were 85-fold (M2), 165-fold (M3), and 191fold (M4) greater, respectively. The Mn content in soil from reference site M1 was similar to the background value.
Table 2 Correlation analysis of ShannoneWeiner index and soil physicochemical properties.
H TMn EMn pH SOM TP EP TN EN
H
TMn
EMn
pH
SOM
TP
EP
TN
EN
1 0.973** 0.911** 0.288 0.568 0.277 0.048 0.993** 0.959**
1 0.976** 0.237 0.507 0.143 0.077 0.951** 0.897**
1 0.095 0.401 0.064 0.002 0.877** 0.811**
1 0.675* 0.056 0.704* 0.317 0.259
1 0.154 0.348 0.588* 0.537
1 0.311 0.344 0.341
1 0.012 0.029
1 0.976**
1
**Denotes significant correlation at p < 0.01. *Denotes significant correlation at p < 0.05. Abbreviations: H, ShannoneWeiner index; TMn, total Mn concentration; EMn, extractable Mn concentration; SOM, soil organic matter; TP, total P; EP, extractable P; TN, total N; EN, extractable N.
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3.2. DGGE analysis
Table 3 GenBank homologies for sequences from recovered DGGE bands.
Nested PCR amplifications were successful, and endophytic bacterial communities associated with different leaf samples were observed on the DGGE gel (Fig. 1). Overall, the band number, intensity, and composition in various sites showed significant differences. Some bands, such as 7, 8, 9, 10, and 17, were detected in all sites. Bands 1 and 5 were present only in site M1. Bands 2, 3, 6, 14, and 15 were not detected in sites M3 and M4, while band 11 was not detected in sites M1 and M2. Site M4 contained some unique bands, such as 12 and 13, that were absent in other sites. Meanwhile, the common bands from site M1 were more intense than from other sites. The ShannoneWeiner index was calculated based on the proportional intensity of bands. Community M1 had the highest diversity index, followed by M2, M3, and then M4 (Fig. 2). The ShannoneWeiner index between various sites was significantly different. Correlation analysis (Table 2) showed that the ShannoneWeiner index was significantly negatively correlated with total soil Mn concentration and extractable Mn concentration (p < 0.01), and significantly positively correlated with total soil nitrogen concentration and extractable nitrogen concentration (p < 0.01). PCA showed that the first two components explained 81.6% of the variance in the endophytic bacterial community. PC1 and PC2 explained 44.1% and 37.4% of the total variance, respectively. Endophytic bacterial communities from four sites were distinctly clustered into four groups, with M1 in the second quadrant, M2 in the third quadrant, M3 in the first quadrant, and M4 in the fourth quadrant (Fig. 3).
Band name
Genbank The nearest BLAST matches accession no.
WY WY WY WY WY WY WY WY WY WY WY WY WY WY
KC410811 KC410814 KC410818 KC410820 KC410823 KC410826 KC410829 KC410832 KC410835 KC410839 KC410841 KC410844 KC410848 KC410850
1-1 2-1 3-3 4-1 5-1 6-1 7-1 8-1 9-1 10-2 11-1 12-1 13-2 14-2
WY 15-2 KC410853 WY 16-1 KC410856 WY 17-2 KC410859 WY 18-1 KC410862
Similarity %
Uncultured Syntrophus sp (JN392959) 98% Sphingopyxis sp (JQ658411) 99% Pseudomonas putida (JX401461) 99% Uncultured delta proteobacterium (HM438022) 98% Uncultured Bacteroidetes bacterium (JX240980) 100% Uncultured Ohtaekwangia sp (JQ771971) 97% Uncultured Actinomyces sp (AY435193) 99% Uncultured Chloroflexi bacterium (JN038250) 99% Filobacillus milosensis (NR027209) 99% Alkalilimnicola ehrlichii (CP000453) 98% Marinobacter lipolyticus (JN202615) 94% Lysobacter spongiicola (NR041587) 95% Uncultured bacterium (HQ190505) 97% Uncultured gamma proteobacterium 98% (JF727680) Uncultured Gemmatimonadetes bacterium 97% (AY795668) Salinisphaera sp (AB735546) 96% Micrococcus yunnanensis (JX885670) 100% Uncultured Acidobacteria bacterium 99% (HM447889)
statistical analysis (Fig. 5) showed that bands 1, 2, 3, 4, 10, 11, 12, 14, and 16 belong to Proteobacteria, bands 5 and 6 belong to Bacteroidetes, bands 7 and 17 belong to Actinobacteria, and all others belong to distinct groups, respectively. The sequencing results and their phylogenetic relationship suggested that endophytic bacterial species in the leaves of P. americana were abundant and diverse.
3.3. Sequence and phylogenetic analysis 4. Discussion Eighteen bands of interest were excised from the DGGE gel for cloning and sequencing (Table 3). A phylogenetic reconstruction of these cloned sequences, together with sequences from their closest relatives from the GenBank database, is shown in Fig. 4. Further
Studies of the composition of endophytes of hyperaccumulating plants living in a contaminated environment have attracted much attention because of their biotechnological applications in
Fig. 3. Principal component analysis of endophytic bacterial communities generated from DGGE band patterns.
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Fig. 4. Neighbor-joining phylogenetic tree of partial 16S rRNA gene sequences amplified from samples of leaves from P. americana. Bootstrap support values with 1000 replicates are given along the branches.
bioremediation [17,29]. Many metal-resistant endophytic bacteria have been isolated from various hyperaccumulators, such as Elsholtzia splendens (copper), Alyssum bertolonii (nickel), and Solanum nigrum L. (cadmium) [4,26,44]. However, little attention has been paid to the composition of endophytic bacterial communities of Mn-hyperaccumulators growing naturally on Mn mine sites. In this study, we explored the diversity of the endophytic bacterial community associated with the Mn-hyperaccumulator P. americana in an Mn mine by a culture-independent approach. The sample sites were highly polluted by Mn (Table 1), However, P. americana grew well and endophytic bacteria were detected in its tissues, which suggested they had adapted to such heavy metal stress through coevolution over an extended period. The endophytic
bacteria found in this experiment are likely to have high Mn tolerance, and might play an important role in improving P. americana growth and health under significant Mn stress. The ShannoneWeiner index decreased significantly with the increasing Mn concentrations from M1 to M4 (Fig. 2). Meanwhile, correlation analysis showed that the ShannoneWeiner index was significantly negatively correlated with total soil Mn concentration and extractable Mn concentration at p < 0.01 (Table 2). Further PCA analysis showed that endophytic bacterial communities from the four sites scattered in four different quadrants, indicating that the community structure of the endophytic bacteria had changed markedly. Heavy metal pollution can change the size, composition, and activity of plant-associated microbial communities [14,52].
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Fig. 5. Distribution of the bacterial category of endophytic bacterial 16S rDNA clone libraries of P. americana.
Some studies have shown that the pollutant was a dominating factor influencing endophytic bacterial community structure [20]. Our results suggested that the diversity of endophytic bacteria in P. americana at Mn mine sites should be impacted by Mn contamination. Nevertheless, it should be noted that the ShannoneWeiner index was also significantly correlated with soil nitrogen concentration, which indicated soil Mn concentration was not the sole factor influencing endophytic bacterial diversity. A plant’s bacterial endophyte community is thought to be recruited from the rhizosphere, so it is also influenced by the physicochemical properties of the soil [36]. The results of our study suggested soil properties, especially nitrogen, also contributed to the variation of endophytic bacterial diversity in P. americana. A more interesting thing is that nitrogen and Mn concentrations are indeed significantly negatively correlated across the sampling sites. Many studies demonstrated heavy metal pollution can influence soil nitrogen contents and chemical forms through inhibiting nitrogen mineralization, nitrification, denitrification and biological nitrogen fixation [33,51]. In this study, nitrogen concentrations decreased with the increasing Mn concentrations which indicated long term Mn pollution may influence soil nitrogen concentration and chemical forms, and further influence endophytic bacterial diversity. In general, the factors influencing endophytic bacterial diversity are varied and their interactions are complex, especially in the field. Therefore, further research is needed to find the exact relationship between environmental factors and endophytic bacteria of P. americana. It is well known that the bacteria isolated from polluted environments are more tolerant to higher concentrations of metals than those isolated from unpolluted areas [18,42]. Further, metal tolerance of bacterial communities increases with the addition of metals, by the death of sensitive species and subsequent competition and adaptation of surviving bacteria [10,39]. Some DGGE bands in this study disappeared with the increasing Mn concentrations from sites M1 to M4, while other bands appeared at the higher Mn concentrations. These unique endophytic bacteria sequence types were intriguing. The sequence types found exclusively in site M1 (such as bands 1 and 5) may represent Mnsensitive endophytic bacterial species. Bands that disappeared in sites M3 and M4 (such as bands 3 and 14) represent species that can only endure relatively light Mn pollution, while the sequence types found exclusively in sites M3 and M4 (such as bands 11, 12, and 13) may be indicative of highly tolerant endophytic bacterial species. As previously mentioned, the influence mechanisms of Mn pollution on these endophytic bacterial species are complex. One possible mechanism is that Mn influence endophytic bacterial species through impacting soil nitrogen concentration and chemical forms
indirectly. Furthermore, band 12 belonged to Lysobacter, which can produce an antibiotic and suppress plant pathogens [19,22]. Band 13 was related to an uncultured bacterial species, implying that it might be a novel genera that is emerging through extensive coevolution with P. americana. Interactions between P. americana and these special endophytes may be important for survival and growth of plants in highly Mn-contaminated soils. The highly tolerant endophytic bacterial species associated with P. americana may have extensive application in improving phytoremediation efficiency in Mn-contaminated soils [6,44]. One question that needs to be explored is only 3 samples from one uncontaminated soil were analyzed which is limited for comparison researching. Some special species may cannot be discovered as this reason. Thus, it is worthwhile to conducted more extensive investigation in future work. Different hyperaccumulators can harbor different endophytic bacterial populations. Chen analyzed the diversity of bacterial endophytes associated with Cd-hyperaccumulator S. nigrum L. growing in mine tailing. Sphingomonas and Pseudomonas were found to be dominant genera of S. nigrum L. [7]. In this study, phylogenetic analysis revealed that the majority of clones were affiliated with Proteobacteria (50%, nine bands), which included g (33.3%, six bands), d (11.1%, two bands), and a (5.6%, one band) subclasses. The sequences of other clones showed they belong to Bacteroidetes (11.1%, two bands), Actinobacteria (11.1%, two bands), Chloroflexi (5.6%, one band), Bacilli (5.6%, one band), Gemmatimonadetes (5.6%, one band), and Acidobacteria (5.6%, one band), respectively. In addition, 5.6% (one band) of the sequences was unclassified, but showed high similarity to uncultured bacterial sequences (Fig. 4). Our results demonstrated that Proteobacteria may be the dominant endophytic bacterial genus of P. americana. Proteobacteria also make up the largest fraction of the clone library of other endophytic bacterial communities [47]. Further, the biological functions of Proteobacteria are varied, including growth promotion, pollutant degradation, and assistance with phytoremediation of heavy metals [30]. 5. Conclusions All of the endophytic bacteria detected in the current study were classified into 10 different divisions, suggesting that endophytic bacterial species of P. americana are abundant and diverse. The phylogenetic analysis revealed that different species occupied remarkably different genetic positions, which might have different implications for their performance under Mn-contaminated conditions. Our future work will be to isolate endophytic bacterial strains from P. americana and cultivate them independently to investigate their effects on phytoremediation under controlled conditions. Acknowledgments This study was supported by grants from the National Natural Science Foundation of China (41271338, 41303066) and the Special Environmental Protection Foundation for Public Welfare Project (201009032) and the National Science Foundation for Post-doctoral Scientists of China (2013M530685) and the State Key Laboratory Program, Chinese Research Academy of Environmental Science (SKLECRA2013OFP03). Special thanks to Professor Zeng (Hunan Agriculture University) for his assistance in the sampling process. References [1] E.R. Alford, E.A.H. Pilon-Smits, M.W. Paschke, Metallophytes: a view from the rhizosphere, Plant Soil 337 (2010) 33e50.
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