Characterization of bacterial communities associated with the exotic and heavy metal tolerant wetland plant Spartina alterniflora

Heavy metal pollution has seriously disrupted eco-balance and transformed estuaries into sewage depots. Quanzhou bay is a typical heavy metal-contaminated estuary, in which Spartina alterniflora has widely invaded. Plant-associated microbial communities are crucial for biogeochemical cycles, studies of which would be helpful to demonstrate the invasion mechanisms of plants. Meanwhile, they are indispensable to phytoremediation by enhancing the heavy metal tolerance of plants, facilitating heavy metal absorption rate and promoting growth of plants. In the present study, S. alterniflora-associated rhizo- and endobacterial communities from 3 experimental sites were investigated by 454-pyrosequencing. Heavy metal screening generated 16 culturable isolates, further biochemical assays suggested these clones possess various abilities such as phosphate solubilization, indole-3-acetic acid (IAA) production and 1-aminocyclopropane-1-carboxylate (ACC) deaminase production to accelerate heavy metal uptake and growth of the host. This study revealed the bacterial community structures and characterized the predominant resident bacterial strains of S. alterniflora-associated rhizo- and endobacteria under heavy metal stress, and isolated several bacterial species with potential ecological function.


Results
Differences of microbial diversity between rhizosphere soil and roots of S. alterniflora. The heavy metal content of sediments and rhizosphere soil was assessed. The results showed that there are different degrees of heavy metal pollution in the 3 sampling sites (see Supplementary Table S1 online). To investigate the rhizo-and endobacterial diversity associated with S. alterniflora under different degrees of heavy metal contamination, we applied 454-pyrosequencing targeting V4 region of 16S rRNA. The 454-pyrosequencing generated over 155,346 reads. In total, 143,009 reads were obtained after quality control (average 29,403 reads per rhizosphere sample and 16,309 reads per endophytic sample). Trimmed sequences generated 14,537 operational taxonomic units (OTUs). On average, 4629 and 1191 OTUs per rhizosphere and endophytic sample were detected, respectively. Based on the rarefaction data (see Supplementary Fig. S1 online) and diversity indices of 454 tag sequences (Table 1), the sequencing depth could not completely cover full diversity of rhizosphere soil, indicating that the microbial community of rhizosphere soil was more diverse than that of the plant roots. It was worth noting that S3 hosted the most diverse and least abundant rhizobacterial community, while R3 displayed a reversed pattern. Weighted PCoA plot ( Fig. 1) showed that R1/R2 and S1/S2 form clusters respectively, while S3/R3 was distant. The results suggested that both rhizo-and endobacterial communities were specific in geographical location.
Composition of the rhizo-and endobacterial communities. The composition of rhizo-and endobacterial communities was highly distinct (see Supplementary Fig. S2 online), and the phyla and genera (partial) with significant differences in relative abundance in rhizosphere soil and roots were shown in Fig. 2. The relative abundance of Actinobacteria and Firmicutes in root was significantly higher than that in rhizosphere soil. Chloroflexi constituted the majority of sequences in rhizosphere soil but showed less abundance in endophytic root. Acidobacteria was the second abundant phylum in both samples. The third abundant phylum allocated to Proteobacteria (12.69 ± 0.58% in soil, 9.66 ± 6.13% in root). Surprisingly, Actinobacteria seemed to concentrate Differences of rhizo-and endobacterial community structure. For a better understanding of rhizoand endobacterial communities, we screened out 8 phyla and 24 genera that had relative abundance > 1% in at least one site (Figs. 3, 4). Overall, rhizobacterial communities (S1, S2 and S3) were dominated by Chloroflexi. Acidobacteria, Actinobacteria and Fusobacteria were found enriched in S3 compared to S1 and S2. Actinobac-   16S rRNA sequencing. By using the same environmental DNA extracts and clones obtained from traditional cultivation method, small-scale verification conducted in lab by full-length 16S rRNA sequencing revealed a slightly different pattern. Taxonomic characterization suggested that most of the isolates were Firmicutes (in soil and endophytic leaves) and Proteobacteria (in endophytic roots). Results turned out to be the subsets of 454-pyrosequencing dataset, indicating 454-pyrosequencing an efficient way to get better insight of microbial community structure since large proportion of bacteria could be underrepresented due to disadvantages of traditional methods. But several genera like Xanthobacter, Methanoplanus, Exiguobacterium, Rheinheimera, Sinorhizobium, Yokenella, Planococcus, etc. that were believed to possess multiple potential ecological functions  www.nature.com/scientificreports/ were identified by full-length sequencing but showed no representation in 454-pyrosequencing (see Supplementary Tables S2 and S3 online). Sequences with low abundance might be underrepresented by high-throughput methods. Therefore, combination of traditional and next-generation sequencing strategies would provide us a comprehensive understanding of microbiology community under natural status.

Identification and biochemical characterization of heavy metal resistant clones. Refer to pre-
vious studies on heavy metal content of wetland plants and preliminary experiments, proper concentrations were added for screening of heavy metal resistant endophytes (50 ppm Cu 2+ , 100 ppm Pb 2+ , 50 ppm Zn 2+ , 5 ppm Cd 2+ , 5 ppm Cr 6+ and 30 ppm Ni 2+ ). Most of the screened endophytes showed multiple resistances to different heavy metal, 5 isolates of them were able to grow on non-nitrogen medium. Biochemical characterization resulted in thirteen IAA producers, five ACC deaminase producer, one siderophore producer and one isolate that could solubilize phosphate (Table. 2). Blastn analysis of these sequences revealed that 34 of them were very similar to Psychrobacter sp. PRwf-1 (NR_074709), this isolate could tolerate 3 out of 5 examined heavy metal and also produce IAA, ACC deaminase. The second highest hits could be affiliated to Lysinibacillus fusiformis strain DSM 2898.

Discussion
Quanzhou bay is reported to have varying degrees of heavy metal contaminations. Despite the adverse environmental impacts, S. alterniflora still thrives and occupies dominant niche. A worldwide investigation indicated that wetlands soil consist of Proteobacteria, Bacteroidetes, Acidobacteria, Firmicutes and Actinobacteria as the five major phyla 22 . Meanwhile, a previous study also shows that Proteobacteria, Bacteroidetes, Chloroflexi and Firmicutes are major phyla of rhizoplane bacteria in S. alterniflora monoculture 18 . Based on our study, Chloroflexi (47.20%) was the most abundant phylum in rhizosphere soil. Its relative abundance was higher than the Chloroflexi recovered from the other heavily contaminated sediment samples 23 . Chloroflexi is generally found in intertidal sediment and moderately acidic wetland 24,25 , and is believed to be associated with nitrite-oxidizing 26 , biological nutrient removal (BNR) processes 27 , sediment carbon cycling 28 , reductive dehalogenation of polychlorinated biphenyls and organohalide-respiring 29,30 , etc. Approximately 9% of OTUs were allocated to six classes in Proteobacteria. Among them, the Delta-, gammaand betaproteobacteria together accounted for 85.3% of proteobacterial OTUs. It is documented that Deltaproteobacteria is a major group of sulfur-reducing bacteria and the major class of phylum Proteobacteria in the rhizosphere of mangrove 3,31 . Endophytic roots of monoculture S. alterniflora are predominated by Proteobacteria, Cyanobacteria, Bacteroidetes, Firmicutes and Spirochaetes 18 . Our data show that although Actinobacteria is sensitive to Cd, Zn contamination 32 , it formed the largest phylum (39.27%) in endobacterial community. Actinobacteria have been studied as soil bacteria occur abundantly in most plants. They are thought to be indispensable in organic material decomposition, and were revealed as endophytes in endophytic roots of S. alterniflora in the present study. Acidothermus was the most frequently observed genus, followed by Acidobacterium (13.5%), Demequina (5.3%) and sulfate-reducing bacteria Desulfovibrio. Members in these genus are believed to play important roles in the metabolism of nitrogen, phosphorus, sulfur and some other organic compounds in wetland system 33 . Above all, both rhizo-and endobacterial community compositions were distinct from previous studies on that of S. alterniflora under normal growing environment 34,35 . These interesting differences observed in our study might be the results of the heavy metal stress on S. alterniflora in situ. Site 3 was located at an oil terminal, the heavy metal content assessment revealed that rhizosphere soil collected from site 3 showed the highest concentration of Cr (see Supplementary Table S1 online). As shown in PCoA plot, both rhizo-and endobacterial data at site 3 were distinguished with those from other sites. Comprehensive comparison of bacterial potential ecological functions and their abundance in each site revealed a relative higher abundance of ecological functional bacteria at site 3 (see Supplementary Table S4 online). Previous studies demonstrated that high richness and abundance of sulfate-reducing bacteria (SRB) occurred in rhizosphere soil of S. alterniflora during the late growing season, suggesting that the abundant SRB might have close relationships with decomposition of soil organic matters produced by S. alterniflora 36 . Notably, microorganisms involved in sulfur cycle had significantly higher abundance in rhizosphere soil (1.48%) and root (11%) collected from site 3 compared to that collected from site 1 and 2. These sulfur cycle participants (mainly associated with sulfatereduction) might also possess ability to oxidize acetate or other organic compounds 37 , and are considered as numerically important members on macrophyte root surfaces 38 . Those root-associated bacteria of S. alterniflora were great contributors to sulfur accumulation in S. alterniflora-invaded stands and could cause the higher sulfur concentration in situ than that of native plants or unvegetated zones 39 .
Other bacteria groups possess potential ecological functions such as phosphate solubilization, biodegradation, aromatic compound degradation, crude-oil degradation etc., shared the same pattern. Commonly, Anaerolineae is often recognized as a large component of microbial communities in sludge wastewater treatment plants 40 , and has been known to be associated with anaerobic degradation of oil-related compounds. Inconsistent with the previous reports, Anaerolineae was found constituted of over 18% of R3 but could barely found in R1 and R2. Interestingly, its abundance in rhizosphere soil appeared to be the lowest at site 3, indicating that S. alterniflora might accumulate bacteria that possess ecological functional to help its survival under various environment stresses.
S. alterniflora could enrich a certain amount of heavy metals. Screening of heavy metal resistant bacteria resulted in 16 different endophytes that showed resistance against at least one of the tested heavy metal ions. Some of the identified endophytes are previously studied as functional bacteria for phytoremediation. For example, a clone (38.82% of leave endophytes) showed resistance against three different heavy metal ions (Cu 2+ , Pb 2+ , Cr 6+ ) was allocated to genus Psychrobacter, members of which are suggested to be applied in phytoextraction 41 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Lysinibacillus fusiformis strain had the second abundant hits (14.29%), which is proved to have potentials for plant growth promotion due to their abilities to resist/reduce chromate at high level and resist/accumulate boron [43][44][45] . Nitrogen (N 2 )-fixing bacteria are able to form symbiotic association with various plants 46 . This functional type of bacteria (e.g. Azospirillum, Azotobacter, Oceanomonas) have been isolated from rhizosphere and proved to have significant impact on the nitrogen cycle of the wetland ecosystem. Endophytic N 2 -fixing bacteria likely constitute only a small percentage of total endophytic bacteria, and the increase of the endophytic N 2 -fixing bacteria population has been considered as possible way for plants to increase nitrogen fixation 47,48 . Endophytic N 2 -fixing bacteria were found in previous researches: a diversity of Azoarcus spp. has been recovered from Kallar grass 49 ; and Klebsiella sp. strain Kp342 have been proved to fix N 2 in wheat 50 . Likewise, N 2 -fixing endophytes seem to relieve nitrogen deficiencies of sweet potato in nitrogen-poor soils 51 . Moreover, some members of Paenibacillus are found to be N 2 -fixing bacteria 52 . In this case, we inoculated all endophytic isolations on non-nitrogen medium and obtained microbes from Paenibacillus, Lysinibacillus, and Chryseobacterium. Paenibacillus were identified in both leaves and roots, indicating that they might promote plant growth through fixing atmospheric nitrogen and dissolving phosphate.
Bacterial endophytes could promote plant growth by a number of different mechanisms, such as production of phytohormones 53 , phosphate solubilization activity 54,55 , nitrogen fixation 56 , siderophore biosynthesis 57,58 , and providing essential nutrients to host plants 59 . Like PGPR, endophytes can also promote plant growth by expressing ACC deaminase. In addition, the resistance of plants treated with ACC deaminase-containing PGPR to flood and heavy metal stress is significantly enhanced 60,61 . IAA, a plant hormone that does not apparently function as a hormone in bacterial cells, may have evolved in bacteria due to its importance in bacterium-plant relationship. In biochemical characterization of the isolated heavy metal-resistant clones, 13 IAA-producing isolates were identified. Among them, 5 isolates showed positive reaction in ACC-deaminase assay. The 16S rRNA sequencing results suggested that 5 ACC deaminase-containing endophytes were from Psychrobacter, Lysinibacillus and Bacillus. In addition, these isolates showed high levels of IAA synthesis. These results indicated that S. alterniflora was colonized by various kinds of endophytes. Some of the endophytes could tolerant a certain concentration of heavy metal and produce bacterial products to satisfy self-survival, improve plant tolerance to heavy metals and promote plant growth. To better understand how the rhizo-and endobacteria contribute to the heavy metal resistance of S. alterniflora, high-throughput sequencing assay focusing on functional genes is warranted for further study.
In conclusion, sediment of Quanzhou bay was contaminated with various degrees of heavy metals. This study contributes to our understanding of the composition and potential ecological functions of the rhizo-and endobacteria associated with S. alterniflora. The overall pattern of both rhizo-and endobacterial community structures were different from that reported in previous studies. The site 3 was located at an oil terminal with high level of Cr contamination. The rhizobacterial diversity decreased at site 3, root endophytes evolved to higher diversity with a large proportion of ecological functional bacteria for heavy metal accumulations, host plant growth promotion and crude-oil degradation. Some comments have suggested that S. alterniflora could alter the community structure of related functional microorganisms, even affect the carbon, nitrogen, and sulfur cycles in habitat 62,63 . Based on the analysis of datasets in the present study, root-associated bacteria of S. alterniflora might have the potential to affect nutrient metabolism in wetland ecosystem, especially nitrogen, phosphate, sulfur and carbon cycles. Culture-dependent method together with biochemical assay revealed that endophytes could tolerant certain concentration of heavy metals. Meanwhile, they could act through nitrogenfixing, phosphate-solubilizing, IAA-producing and ACC-deaminase producing to participate in energy cycles and promote plant growth. Further investigation on the data indicated a considerable proportion of microorganisms with the potential to be applied in phytoremediation and natural medicine development. The functions of these microbial communities need to be further studied so as to elucidate the mechanism of S. alterniflora invasion and survival under heavy metal stress.

Materials and methods
Sample collection. Plants, rhizosphere soil and sediment samples were sampled from three sites of wetland in Quanzhou bay, Fujian, PR China in Dec 4, 2012. Sampling site 1 is located at a sluice of a residential quarter (N24°52.493′, E118°36.764′), site 2 was located at the north-west coast of Jinjiang bridge (N24°52.603′, E118°37.635′) and site 3 is at Houzhu oil terminal (N24°52.655′, E118°40.936′). All subsequent handling of samples was treated with sterilized gloves or tools. Plants with rhizosphere soil were immediately sealed in polyethylene bags and transported to the laboratory in transportable cooler (4 °C). Fresh samples were sorted as rhizosphere soil (S), roots (R) and leaves (L).

Sample pretreatments.
Rhizosphere soil (S) was carefully removed from the root of the S. alterniflora, then stored at 4 °C. Plant samples were surface sterilized as Idris described 64 . Sterility was checked by blotting plant surface tightly onto tryptic soy agar (TSA) plates and incubating plates at 28 °C for 2 days. Surface-sterilized plant samples were cut into small pieces and classified into leaves (L) and roots (R), then stored at 4 °C.
Screening of heavy metal resistant microorganisms. After pretreatment, fresh rhizosphere soil (2.5 g) and sterilized plant samples (R and L) were soaked in tryptic soy broth and shaken for 2 h at 250 rpm at 28 °C, respectively. The solution was then left without shaking for 1 h to allow the settlement of particles. Various tenfold dilutions were plated on TSA (with 100 mg L −1 cycloheximide), nitrogen-free culture medium and TSA contain different concentrations of CrCl 6 (5-50 ppm), CdCl 2 (2-10 ppm), Pb(NO 3 ) 2 (20-100 ppm), ZnCl 2 (100-500 ppm), CuSO 4 (20-100 ppm), NiSO 4 (10-50 ppm), respectively. Plates were incubated at 28 °C until visible clones were observed. IAA analysis. l-Tryptophan (2.5 mg mL −1 ) was filter sterilized before use. Medium containing 4 mL of nitrogen medium and 1 mL of l-tryptophan (2.5 mg mL −1 ) was inoculated with endophyte clone, and shaken for 4 days at 250 rpm at 28 °C. Removed 1 mL of each suspension and mixed thoroughly with 2 mL Sackowski's reagent 70 , tubes were shielded from light at room temperature for 30 min. Positive reactions with IAA production showed pink color. Standard curve was established with various tenfold dilutions of IAA standard solution measured at 530 nm for absorbance. Endophyte clones were cultured in nitrogen medium with l-tryptophan (0.5 mg mL −1 ) for 48 h before measured for absorbance at 600 nm. Supernatants were mixed with isovolumetric Sackowski's reagent, developed in dark for 30 min, and subjected to measurement for absorbance at 530 nm. Data were recorded for further analysis.
ACC-deaminase production analysis. Endophyte clones were inoculated on DF salts minimal medium respectively 71 , and incubated at 30 °C. Clones that could grow and pass for 5 times were considered able to produce ACC-deaminase. Positive clones were inoculated in 15 mL TSB, shaken for 24 h at 28 °C. Cells were harvested by centrifugation for 10 min at 8000×g at 4 °C, rinsed with DF salts medium for three times. Cells were resuspended with 7.5 mL ADF medium (DF salts medium with ACC final concentration of 3.0 mM), shaken for 24 h at 200 rpm at 30 °C to induce the production of ACC-deaminase. Cells were harvested by centrifugation for 10 min at 8000×g at 4 °C, rinsed with 0.1 M Tris-HCl (pH 7.6) twice. Samples were resuspended in 600 μL 0.1 M Tris-HCl (pH 8.5), added 30 μL methylbenzene and vortexed for 30 s to make crude enzyme. Protein concentration was determined by Bradford protein assay kit (Thermo Fisher) following the manufacturer's instruction. Samples were subjected to ACC-deaminase activity test. Briefly, mixed 200 μL crude enzyme and 20 μL ACC (0.5 M), incubated at 30 °C for 15 min, added 1 mL 0.56 M HCL to terminate the reaction (reaction without crude enzyme and reaction without ACC were set up as control groups). Supernatant (1 mL) was removed to a new tube and mixed with 800 μL HCl (0.56 M) and 300 μL 0.2% 2,4-dinitrophenyl hydrazine (dissolved in 2 M HCl). After incubation at 30 °C for 30 min, samples were mixed thoroughly with 2 mL 2 M NaOH, and