Microbial Ecology https://doi.org/10.1007/s00248-018-1208-y

PLANT MICROBE INTERACTIONS

Different Height Forms of Spartina alterniflora Might Select Their Own Rhizospheric Bacterial Communities in Southern Coast of China Li’an Lin 1 & Wenwen Liu 2 & Manping Zhang 1 & Xiaolan Lin 1 & Yihui Zhang 2 & Yun Tian 1 Received: 30 January 2018 / Accepted: 18 May 2018 # Springer Science+Business Media, LLC, part of Springer Nature 2018

Abstract In the southernmost part of coast of China, two height forms of Spartina alterniflora, tall and short, have invaded Leizhou Peninsula within the last decade. However, the effect of different height forms of Spartina alterniflora on plant–microbe interaction has not been clarified. Here, the community structures of rhizosphere bacteria and the abundance of N- and Scycling functional genes associated with selected S. alterniflora were investigated in the field and a common garden. The community structure of tall-form S. alterniflora was distinct from short-form S. alterniflora at OTU level in the field, even after transplantation into a common garden. The abundance of bacterial amoA, nirS, and nosZ in tall S. alterniflora was significantly greater than those in short S. alterniflora in the field; however, this difference disappeared in a 1-year common garden experiment. These results suggested that compared with the tall-form S. alterniflora, the rhizosphere of short-form S. alterniflora harbored fewer nitrification–denitrification related microorganisms, which might benefit from conserving N in an N limited habitat. Together, our results suggested that tall- and short-form S. alterniflora can host their specific rhizosphere microbial communities and had different strategies of N usage via selecting the composition of rhizosphere bacterial assemblages, which in turn might determine the growth and invasiveness of S. alterniflora in its introduced range. Keywords Spartina alterniflora . Rhizosphere bacteria . Plant–microbe interaction . Nitrogen cycling

Introduction The microbial community colonizing the rhizospheric environment contributes to plant growth, productivity, nutrition, diseases, and plant restoration [1]. In return, plants secrete a carbon source into their direct surroundings and feed the microbial community, influencing their composition and activities [2]. Stable isotope probing and a DGGE community

Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00248-018-1208-y) contains supplementary material, which is available to authorized users. * Yihui Zhang [email protected] * Yun Tian [email protected] 1

Key Laboratory of the Ministry of Education for Coastal and Wetland Ecosystems, School of Life Sciences, Xiamen University, Xiamen 361102, China

2

College of the Environment and Ecology, Xiamen University, Xiamen 361102, China

experiment demonstrated that exudate-consuming bacterial rhizosphere populations were more distinct than populations that did not utilize the root exudates [3]. Plant roots can also inhibit growth of specific rhizosphere microbes through secreting secondary metabolites [4, 5]. As a pioneer species, Spartina alterniflora exhibits a complex interplay between sediment and microorganisms in coastal ecosystems, which typically plays a pivotal role in the dynamics of nutrients in the respective C, N, and S cycles. S. alterniflora invasion changed the functional microbial community and increased N-fixation rates in an invaded zone [6]. In addition, S. alterniflora invasion into mangrove habitats could reduce N2O effluxes under exogenous N loading [7]. Spartina alterniflora, which is a perennial salt marsh grass, was introduced to China in 1979. To date, S. alterniflora has been widely distributed over 19 latitudinal degrees across East and South China [8, 9], representing the largest Spartina invasion in the world [10]. Owing to its extensive expansion, S. alterniflora has caused a number of ecological impacts on native ecosystems [11, 12]. Leizhou Peninsula, the third largest Chinese peninsula with an area of about 8500 km2, is located on the southernmost part of Guangdong province.

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According to our field investigation, we found that two distinct height forms, tall-form S. alterniflora (over 1 m in height, about 160 tillers/m2) and short-form S. alterniflora (about 50 cm in height, about 300 tillers/m2), exist in Leizhou Peninsula. Here we report the discovery and spread of a distinctly short-form Spartina alterniflora in Leizhou Peninsula, and as far as we know, a similar dwarf Spartina has not been reported elsewhere on the Chinese coast. A Bshort form^ of S. alterniflora has been reported from the species’ native range and found commonly on the Atlantic and Gulf coasts. Much debate has focused on whether this short form is environmentally induced or another genotype invasion [13]. Tall and short forms of S. alterniflora appear to be genetically different based on a 9-year common garden study [13]. Long-term waterlogging may create a highly reducing environment, which can result in limited root oxygen as well as sulfide enrichment, ultimately reducing S. alterniflora N uptake and assimilation and, hence, growth [14]. A dwarf ecotype of Spartina is reported which differs significantly from the previously reported short-form S. alterniflora, with higher shoot density and less diameter than the short-form S. alterniflora, which invaded San Francisco, and the dwarf condition is not caused by endophytic fungi [13]. To our knowledge, little attention has been paid to the rhizosphere bacterial composition and element cycling for both tall- and short-form S. alterniflora. In recent years, next-generation sequencing techniques have been widely applied in the study of microbial ecology in various environment matrices, which can provide more detail of the microbial community [15, 16]. We hypothesized that different height forms of S. alterniflora have distinct rhizosphere bacterial community structure even in the same sediment environment and have different strategies of N or S usage in N-limited and S-rich sediments. In order to test this hypothesis, high-throughput sequencing of 16S rRNA gene amplicons and real-time quantitative polymerase chain reaction (qPCR) assay were employed to describe the bacterial assemblages and the abundance of N- and S-cycling functional genes associated with tall and short growth form S. alterniflora in the field and common garden. Our work should ultimately help in better understanding the mechanism that different height forms of S. alterniflora select their own rhizospheric bacterial communities to respond the characteristic of habitat, which may contribute to the growth and invasiveness of S. alterniflora.

2015 (20° 55′ N, 110° 9′ E). In the sampling field site, tallform S. alterniflora (over 1 m in height) and short-form S. alterniflora (about 50 cm in height) were present and invaded the mudflat zone, each forming a monoculture stand. Replicate tall-form S. alterniflora (W_TR, n = 5) and shortform S. alterniflora (W_SR, n = 5) rhizosphere sediment samples were collected from a 10-cm depth. Two bulk sediment control samples from the unvegetated mudflat (MF) were collected. For rhizosphere sediment analysis, the roots of S. alterniflora were dug up and shaken gently and then rinsed with 0.9% NaCl solution to remove the root-adhering sediments [17]. After collection into sterile plastic bags, the samples were stored and transported to the laboratory as soon as possible at 4 °C. Then, the samples were divided into two parts, one part was stored at 4 °C for physicochemical analyses and the residual samples were stored at − 80 °C for molecular experiments.

Common Garden Experiment To ascertain the difference of rhizosphere bacterial community organization and function of field tall- and shortform S. alterniflora under no nutrition limit condition, we conducted a common garden experiment in a greenhouse at the Xiang’an campus of Xiamen University (24° 37′ N, 118° 18′ E). In March 2015, rhizomes of tall- and short-form S. alterniflora clones were collected using a corer (8-cm diameter to 20-cm depth, n = 5 per Spartina type) and stored in plastic bags for transport to the greenhouse. Replicate rhizome fragments (n = 5) were planted individually in pots (18 cm diameter, 24 cm deep) and placed in a plastic pool, such that the pool contained plants from each of the five populations of tall- and short-form S. alterniflora clones. Each pot was filled with mixture of 50% nutrient soil and 50% vermiculite (by volume). Plants were cultured with 10 PSU sea water, maintained at constant salinity by adding fresh water every other day, and initially fertilized with 0.5 g per plant of 15-15-15 Plantex fertilizer (Jiffy, Norway), which was added to the irrigation sea water at the beginning of the greenhouse experiment. In November 2016, the rhizosphere soil samples were collected from tallform S. alterniflora (C_TR) and short-form S. alterniflora (C_SR) for the downstream molecular experiment. After one more year common garden experiment, the height form still remained distinct.

Materials and Methods Physical and Chemical Parameters Analyses Site Description and Soil Sample Processing Sediment samples were collected along the tidal wetland in Leizhou Peninsula of Guangdong province, China, in August

Nitrate (NO3−) and nitrite (NO2−) were extracted from the sediment with 2 M KCl, followed by filtration and measurement using an AA3 nutrient automatic analyzer

Different Height Forms of Spartina alterniflora Might Select Their Own Rhizospheric Bacterial Communities...

(Bran-Luebbe, Germany). The extracted ammonium was measured by Tir-223 (Bran-Luebbe, Germany). The total N content, organic C concentration, and total S content were determined using a CN elemental analyzer (Elementary, Germany).

data collection, and data analysis were carried out with the Bio-Rad CFX96 qPCR instrument following the manufacturer’s protocols.

Data Processing and Statistical Analyses DNA Extraction, PCR, and Illumina Amplicon Sequencing DNAwas extracted from 0.5 g of soil using a Power Soil DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, CA, USA), in accordance with the manufacturer’s protocol. The extracted DNA was quantified using a NanoDrop spectrophotometer 2000 (Thermo Scientific, USA). The V3-V4 region of the bacterial 16S ribosomal RNA gene was amplified using PCR (95 °C for 3 min, followed by 27 cycles at 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 45 s and a final extension at 72 °C for 10 min) using the primers 338F 5′-barcodeA C T C C TA C G G G A G G C A G C A - 3 ′ a n d 8 0 6 R 5 ’ GGACTACHVGGGTWTCTAAT-3′, where the barcode is an eight-base sequence unique to each sample. PCR reactions in an ABI GeneAmp® 9700 (ABI, USA) were performed in triplicate 20 μL mixtures containing 4 μL of 5 × FastPfu buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of each primer (5 μM), 0.4 μL of FastPfu Polymerase, and 10 ng of template DNA. Amplicons were extracted from 2% agarose gels and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) following the manufacturer’s instructions and quantified using QuantiFluor™-ST (Promega, USA) and submitted to MJB (Shanghai Majorbio Bio-Pharm Tech, China) for pair-end sequencing (TruSeq™DNA Sample Prep Kit, USA) on the Illumina MiSeq PE300 platform with the MiSeq Reagent Kit v3 (Illumina, USA).

Real-Time Quantitative PCR The abundance of 16S rRNA and functional genes was quantified using the SYBR Green I-based qPCR method. Detailed information about the primers used in this study is presented in Table S1. qPCRs and a standard curve analysis were performed using a Bio-Rad CFX96 qPCR instrument (Bio-Rad Laboratories, Hercules, CA, USA). Three independent quantitative PCRs were performed for each gene. Tall- and short-form S. alterniflora had five sediment replicates, and the bulk sediment had two replicates in the field. Tall- and short-form S. alterniflora had five replicates in the common garden experiment. Thermal cycling conditions for the 16S rRNA and functional genes were as published in other studies [18–25]. Standard curves were obtained with serial plasmid dilutions of a known amount of plasmid DNA containing a fragment of the target gene. Thermal cycling, fluorescence

The raw data of the sequences were screened and filtered using QIIME (version 1.7.0) with the following criteria. (i) The 300-bp reads were truncated at any site receiving an average quality score < 20 over a 50-bp sliding window, discarding the truncated reads that were shorter than 50 bp. (ii) Exact barcode matching, allowing two nucleotide mismatch in primer matching, reads containing ambiguous characters were removed. (iii) Only sequences that overlap longer than 10 bp were assembled according to their overlap sequence. Singletons were removed prior to operational units (OTUs) clustering. OTUs were picked at 97% similarity cutoff using UPARSE (version 7.1, http://drive5.com/uparse/), chimeric sequences were identified and removed using UCHIME, and their representative sequences were chosen for alignment and taxonomic assignment [26]. The taxonomy of each representative sequence was analyzed using an RDP Classifier (http://rdp.cme.msu. edu/) against the Silva (SSU123 http://www.arb-silva. de) 16S rRNA database using a confidence threshold of 70%. We used the Shannon community diversity index and OTU number as measures of α-diversity. α-Diversity and β-diversity analyses were conducted based on the OTU table with rarefaction depth 28567. Rhizosphere bacterial community structure of two height forms of S. alterniflora was tested for significance using two-sided Wilcoxon rank-sum test in STAMP (version 2.1.3) [27]. Welch’s inverted was used to calculate confidence interval and Benjamini-Hochberg FDR correction was performed when using Wilcoxon rank-sum test. A nonmetric multidimensional scaling ordination based on the Bray– Curtis similarity matrix using PRIMER (version 5.0) and redundancy analysis (RDA) based on Euclidean distance in Vegan with R (version 3.3.0) was used to investigate differences in the rhizosphere bacterial communities between tall- and short-form S. alterniflora. The analysis of similarities statistic global R was calculated and displayed the degree of the separation between groups of community samples, using permutation/randomization methods on the Bray–Curtis similarity matrix using PRIMER (version 5.0). One-way analysis of variance (ANOVA) followed by the Duncan test in SPSS (version 19.0) was used to analyze the effects of plant type on functional gene abundance, plant height, shoot density, and environmental parameters. Significance was accepted at a level of probability (P) of < 0.05.

Lin L.’a. et al. Table 1 2015 Site

Physicochemical parameters of the samples collected along the tidal wetland in Leizhou Peninsula of Guangdong province, China, in August NH4+(mg/kg)

NO3−(mg/kg)

NO2−(mg/kg)

TN(mg/g)

Org C(mg/g)

TS(mg/g)

W_TR

1.918 ± 0.339

2.748 ± 0.913

0.033 ± 0.005

0.809 ± 0.187

8.400 ± 1.821

1.460 ± 0.205

W_SR MF

2.796 ± 0.739 11.930 ± 0.804

2.431 ± 0.867 1.725 ± 0.006

0.024 ± 0.013 0.026 ± 0.000

0.629 ± 0.087 0.800 ± 0.074

7.849 ± 1.803 9.155 ± 0.068

1.585 ± 0.169 1.060 ± 0.124

NH4+ ammonium, NO3− nitrate, NO2− nitrite, TN total nitrogen, Org C total organic carbon, TS total sulfur, W_TR tall-form Spartina alterniflora rhizosphere sediment samples taken from the field conditions, W_SR short-form Spartina alterniflora rhizosphere sediment samples taken from the field conditions, MF bulk soil taken from the unvegetated mudflat

Nucleotide Sequence Accession Numbers

Results

The raw reads obtained in this study were deposited into the NCBI Sequence Read Archive database (Accession Numbers: SRP094609 and SRP109903).

Plant Height, Shoot Density, and Environmental Parameters Chemical composition and environmental analysis were determined for five tall-form S. alterniflora rhizosphere sediment samples, five short-form S. alterniflora rhizosphere sediment samples, and two bulk sediment samples. The related data concerning these sediment samples is provided in Table 1. Based on physicochemical assessment, the rhizosphere sediment was shown to be limited in N, with low concentrations of NO2− (0.024–0.033 mg/ kg) and NH4+ (1.918–2.796 mg/kg) detected. The ammonium content of bulk soil from the unvegetated mudflat was much higher than that of Spartina rhizosphere sediments. The rhizosphere sediment also showed richness in S in the field, with higher concentrations of the total S (1.460–1.585 mg/g) compared with bulk soil. In general, all other measured chemical components (nitrate, nitrite, total N and organic C) were slightly higher for the tallform S. alterniflora rhizosphere samples (ANOVA, P > 0.05). The most striking characteristic of short-form S. alterniflora was its small size and extremely high shoot density, relative to tall-form S. alterniflora. In the field, stem height of tall-form S. alterniflora was significantly higher than that of short-form S. alterniflora (131 ± 21 vs. 52 ± 13 cm, ANOVA, P < 0.001), but shoot density of tallform S. alterniflora was significantly lower than that of short-form S. alterniflora (167 ± 27 vs. 299 ± 63 tillers/ m 2 , ANOVA, P = 0.005). After transplantation into

Fig. 1 Venn diagram (a). Shannon community diversity index (b). Significant differences in means are indicated by different lowercase letters above bars (ANOVA, P < 0.05). W_TR tall-form Spartina alterniflora rhizosphere sediment samples taken from the field conditions, W_SR short-form Spartina alterniflora rhizosphere sediment samples taken from the field conditions, C_TR tall-form Spartina alterniflora rhizosphere soil samples taken from the common garden with fertilizer addition, C_SR short-form Spartina alterniflora rhizosphere soil samples taken from the common garden with fertilizer addition

Fig. 2 Nonmetric multidimensional scaling (NMDS) ordination for the rhizosphere bacterial community from two height forms of Spartina alterniflora in the field (a), in the common garden (b), and distance box plot both in the field and common garden (c). W_TR tall-form Spartina alterniflora rhizosphere sediment samples taken from the field conditions, W_SR short-form Spartina alterniflora rhizosphere sediment samples taken from the field conditions, C_TR tall-form Spartina alterniflora rhizosphere soil samples taken from the common garden with fertilizer addition, C_SR short-form Spartina alterniflora rhizosphere soil samples taken from the common garden with fertilizer addition

b

Different Height Forms of Spartina alterniflora Might Select Their Own Rhizospheric Bacterial Communities...

A

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C

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common garden plots, the height forms still remained significantly distinct (96 ± 10 vs. 36 ± 14 cm, ANOVA, P < 0.001).

Bacterial Community Diversity and Structure After quality trimming, a total of 706,722 valid sequences were obtained from 20 rhizosphere samples and clustered into 6329 OTUs at equal sequencing depth. The shared OTUs among the samples are shown on a Venn diagram (Fig. 1a). The results showed that 398 OTUs were common for both short- and tallform S. alterniflora in the field and common garden, while there were 102 OTUs unique for tall S. alterniflora in the field (W_TR), 143 for short S. alterniflora in the field (W_SR), 127 for tall S. alterniflora in the common garden (C_TR), and 109 for short S. alterniflora in the common garden (C_SR). In this study, Shannon diversity indices also revealed that bacterial diversity in the common garden was significantly lower than the sample from the field (ANOVA, P < 0.05) (Fig. 1b). This pointed to a notable change in bacterial community diversity and structure after transplantation. However, the difference of rhizosphere bacterial diversity between tall- and short-form S. alterniflora was slight in the field or common garden. At OTU level, as indicated in Fig. 2a, bacterial communities of W_TR and W_SR were significantly distinct from each other (analysis of similarities (ANOSIM), P = 0.009, R = 0.724). The difference between tall- and short-form S. alterniflora in bacterial structure was still present after transplantation (ANOSIM, P = 0.015, R = 0.544) (Fig. 2b). The bacterial communities of four different type rhizosphere samples were significantly distinct from each other (ANOSIM, P = 0.001, R = 0.808) (Fig. 2c). This result supported the hypothesis that bacterial community structure changed Fig. 3 Redundancy analysis triplot of bacterial community structure and soil parameters (NH4+ ammonium, NO3− nitrate, NO2− nitrite, TS total sulfur, TN total nitrogen, Org C total organic carbon). W_TR tall-form Spartina alterniflora rhizosphere sediment samples taken from the field conditions, W_SR shortform Spartina alterniflora rhizosphere sediment samples taken from the field conditions

significantly after transplantation, but the difference in plant height between the two forms of S. alterniflora still remained during the time when they lived in the same environment. Based on the RDA, plant height, sediment NO3−, NO2−, and TN contents had the strongest effect on the bacterial compositions of tall-form S. alterniflora in the field, while the bacterial compositions of short-form S. alterniflora positively correlated with shoot density, sediment NH4+, total S, and total organic C content (Fig. 3). All sequences were classified from phylum to genus, and the compositions of the different rhizosphere samples were similar, but the abundance varied. The abundances of phylum in the sediments are shown in Fig. 4. In Fig. 4a, bacterial phylum across all samples collected from the field were dominated by Proteobacteria (circa 60%), Bacteroidetes (circa 6.7%), Chloroflexi (circa 9.4%), Acidobacteria (circa 3.7%), and Gemmatimonadetes (circa 1.9%). The samples collected from the common garden had a greater abundance of Chloroflexi (circa 17.5%) and Bacteroidetes (circa 12.1%), but fewer Proteobacteria (circa 41%). In addition to the phylum level, bacterial diversity and abundance were also determined more specifically at order level. In the taxonomic units of order, the microbial taxa detected in our samples (Fig. 4b) pointed to a predominance of sulfate reducers among these samples (most importantly Deltaproteobacteria). In the Deltaproteobacteria, our results pointed to predominant members of the sulfate reducers (Desulfobacterales and Desulfuromonadales), together with other groups linked with the biogeochemical S cycle. In the typical order Campylobacterales of the Epsilonproteobacteria, most of the species were microaerophilic. Compared with these samples from the common garden experiment, S. alterniflora in the field were typically characterized by sulfate reducers and

Different Height Forms of Spartina alterniflora Might Select Their Own Rhizospheric Bacterial Communities... Fig. 4 Community bar plot at phylum level (a) and at order level (b). W_TR tall-form Spartina alterniflora rhizosphere sediment samples taken from the field conditions, W_SR shortform Spartina alterniflora rhizosphere sediment samples taken from the field conditions, C_TR tall-form Spartina alterniflora rhizosphere soil samples taken from the common garden with fertilizer addition, C_ SR short-form Spartina alterniflora rhizosphere soil samples taken from the common garden with fertilizer addition

A

B

microaerophile (Fig. 5a), while samples from the common garden were characterized by the Anaerolineales, Rhizobiales, and Nitrosomonadales (Fig. 5b).

Quantification of 16S rRNA, nifH, Bacterial amoA, Archaeal amoA, nirS, nosZ, and dsrB Gene Abundances Both in the Field and in the Common Garden In this work, the abundance of the 16S rRNA gene and functional genes was determined using qPCR to explore the characterization of N and S utilization between talland short-form S. alterniflora. The abundance of nifH in tall-form S. alterniflora presented no significant difference with short-form S. alterniflora but was significantly

greater than that in the bulk soil (Fig. 6a). The copy numbers of nirS, nosZ, and bacterial amoA in W_TR were significantly greater than that in W_SR (Fig. 6a). Therefore, the abundance of denitrification gene in W_TR was significantly greater than that in W_SR. The copy numbers of 16S rRNA, nifH, archaeal amoA, and dsrB genes in W_TR presented no significant difference with W_SR (Figs. 6a and 7a). Thus, we found a distinct functional gene abundance pattern involved in the N-loss and S-reduction between tall- and short-form S. alterniflora. However, the differences of nirS, nosZ, and bacterial amoA gene abundance of the two height forms disappeared in our common garden experiment, although the height forms still remained significantly distinct when living in the same environment (Figs. 6b and 7b).

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Fig. 5 Wilcoxon rank-sum test bar plot of order level in the field (a) and the common garden (b). W_TR tall-form Spartina alterniflora rhizosphere sediment samples taken from the field conditions, W_SR shortform Spartina alterniflora rhizosphere sediment samples taken from the

field conditions, C_TR tall-form Spartina alterniflora rhizosphere soil samples taken from the common garden with fertilizer addition, C_SR short-form Spartina alterniflora rhizosphere soil samples taken from the common garden with fertilizer addition

Discussion

garden, the two different types of Spartina had a different rhizosphere bacterial structure, although bacterial diversity showed no significant difference. The results showed that both tall- and short-form Spartina can select different microorganisms and form their own rhizosphere environment. The RDA results further suggested that the differences in the bacterial community structures of tall- and short-form S. alterniflora rhizosphere were correlated with the N and S contents of the soil. The complex

We investigated the differences of bacterial community diversity and functional genes between tall- and short-form S. alterniflora rhizosphere sediments. Based on the Shannon diversity indices, the short-form S. alterniflora rhizosphere sediment harbored the highest bacterial diversity but showed no significant difference with tall-form S. alterniflora. Both in the field and in the common

Different Height Forms of Spartina alterniflora Might Select Their Own Rhizospheric Bacterial Communities...

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R

Fig. 6 Copy numbers of 16S rRNA and nifH, bacterial amoA, archaeal amoA, nirS, and nosZ genes in the field (a) and the common garden experiment (b). Means and standard errors of soil replicates are plotted. Significant differences in means are indicated by different lowercase letters above bars (ANOVA, P < 0.05). W_TR tall-form Spartina alterniflora rhizosphere sediment samples taken from the field conditions, W_SR short-form Spartina alterniflora rhizosphere sediment samples taken from the field conditions, MF bulk soil taken from the unvegetated mudflat, C_TR tall-form Spartina alterniflora rhizosphere soil samples taken from the common garden with fertilizer addition, C_ SR short-form Spartina alterniflora rhizosphere soil samples taken from the common garden with fertilizer addition

interactions between plants and microbial community are crucial for plant health and growth. Most members of the rhizosphere microbiome play an important role in the part of a complex food web and utilize the large amount of nutrients released by the plant. For example, an overwhelming range of studies indicate that plants have a strong effect on rhizosphere bacterial community composition through different root exudates [3, 28, 29]. Plants may modulate the rhizosphere bacterial community structure to their benefit by selectively stimulating microorganisms with traits that are beneficial to plant growth and health [1]. Given that different rhizosphere environments of two height forms of S. alterniflora may be a major driving force in the regulation of microbial composition on plant roots. Some studies report that invasion by S. alterniflora affects the C, N, and S cycles in the salt marsh ecosystem by altering the community structure of related functional microorganisms [6, 30, 31]. The root itself considered as a more stable niche provides microorganisms with a valuable source of C, such as amino acids, organic acids, and carbohydrates, while rhizosphere microorganisms play the most significant role in the transformation of N compounds (including N fixation, ammonification, nitrification, and denitrification) and help plants to

Fig. 7 Copy numbers of the dsrB gene in the field (a) and the common garden experiment (b). Means and standard errors of soil replicates are plotted. Significant differences in means are indicated by different lowercase letters above bars (ANOVA, P < 0.05). W_TR tall-form Spartina alterniflora rhizosphere sediment samples taken from the field conditions, W_SR short-form Spartina alterniflora rhizosphere sediment

acquire nitrogen, phosphorus, and potassium [1, 32–34]. The role of microbes in N and S cycling in soils can be better understood using the analysis of element cycling marker genes. In our work, the characterization of N cycling and S cycling between tall- and short-form S. alterniflora both in the field and the common garden experiment was discussed. The relatively higher copy numbers of nifH gene in tall S. alterniflora than in the unvegetated mudflat (MF) indicated the effects of the plants on the N fixer community, but the copy numbers of nifH in short S. alterniflora presented no significant difference with tall S. alterniflora in the field. A harvesting experiment indicated that vegetation uptake accounts for only 4–11% of N removal, and 89–96% is due to denitrification in a planted wetland (Lin et al. 2002). Stottmeister et al. [35] also found that rhizosphere microorganisms played a dominant role in organic nutrient mineralization and transformation compared with plants. The influence of height difference S. alterniflora on the copy numbers of bacterial amoA, nirS, and nosZ genes was observed in our work, suggesting the notably different rhizosphere environment (i.e. oxygen concentration, root density and root secretion) (Fig. 6a). Based on our physicochemical assessment, the rhizosphere sediment was shown to be limited in N. A higher activity of nitrification–denitrification has also no benefit to the plants using the available N source in an N-limited habitat. However, the difference between tall-form S. alterniflora with bulk soil was seemingly slight in nitrification–denitrification gene abundance (Fig. 6a). Considering higher denitrification gene abundance in tall-form rather than short-form S. alterniflora, we supposed that the tall-form S. alterniflora in an N-limited habitat tended to favor microorganisms which involved N loss, while the rhizosphere of short-form S. alterniflora favored N conserving microorganisms. Possibly, N availability was the limiting factor to short-form S.

samples taken from the field conditions, MF mudflat, C_TR tall-form Spartina alterniflora rhizosphere soil samples taken from the common garden with fertilizer addition, C_SR short-form Spartina alterniflora rhizosphere soil samples taken from the common garden with fertilizer addition

Different Height Forms of Spartina alterniflora Might Select Their Own Rhizospheric Bacterial Communities...

alterniflora, and therefore, they favored fewer nitrification–denitrification related microorganisms to uptake a limited N source. The difference of denitrification gene abundance disappeared, and the abundance of the nosZ gene in the common garden experiment was obviously greater than that in the field for both tall- and short-form S. alterniflora, possibly due to a sufficient available N source in the nutritional soil used (Fig. 7b). The result seemingly supported the hypothesis that N source might be the limiting factor for Spartina growth, and the tall- and short-form S. alterniflora have different strategies of N (ammonia) usage. Based on our chemical assessment, in addition to its N-limited property, the rhizosphere sediment also showed richness in S in the field. We found that the abundance of sulfate reducers was obviously greater than the abundance of the N cycling gene, accounting for circa 8.559 × 109 and 7.256 × 109 per gram of dry weight in the sediments of tall- and short-form S. alterniflora in the field (Fig. 7). The dsrB gene encoding the dissimilatory sulfate reductase subunit B catalyzes the reduction of sulfite (SO32−) to sulfide (S2−) in SRB (sulfate-reducing bacteria) [36, 37]. Based on dsr gene analysis, the SRB communities are dominated by such families as Desulfobacteraceae and Desulfobulbaceae within the Deltaproteobacteria [31, 38]. Thus, the rhizospheric bacteria community of S. alterniflora might be consistent with the qPCR result of the dsrB gene and make a great contribution to S accumulation in S. alterniflorainvaded stations (Table 1; Fig. 7a). However, the abundance of sulfate reducers in the common garden was lower than in the field samples, possibly due to the environmental difference between the field and the common garden, such as less waterlogging, rich nutrition, and less sulfate (Fig. 7). In community analysis, the percentage of sulfate reducers was also lower than in samples from the field (Fig. 4). Tall- and short-form S. alterniflora plants were transplanted from Leizhou Peninsula to common garden plots in Xiamen city during spring 2015. One year later, plant height still remained distinct for the two forms in the same environment, but the differences of the nitrification– denitrification gene abundance of the two height forms disappeared, although the difference in rhizosphere bacterial structure still remained. This result validated our hypothesis that different height forms of Spartina alterniflora have distinct rhizosphere bacterial community organization and strategies of N usage. In summary, we found that the two distinct S. alterniflora heights showed some significant difference in rhizospheric bacterial community assemblage and the potential different strategy of N usage in an N limited habitat, which in turn might determine the growth and invasiveness of S. alterniflora in its introduced range. Further work is necessary to improve our understanding of the mechanism through more and wider field investigations and more longer-term common garden experiments.

Acknowledgments We thank Professor John Hodgkiss of the City University of Hong Kong for his assistance with the English language. Funding Information This research was supported by the Fundamental Research Funds for the Central Universities (Grant No. 20720150097).

Compliance with Ethical Standards Conflict of Interest The authors declare that they have no conflict of interest. Ethical Approval This article does not contain any studies with human participants or animals performed by any of the authors.

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