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Soil Biology & Biochemistry 68 (2014) 392e401

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Dynamics of the bacterial community structure in the rhizosphere of a maize cultivar

Xiangzhen Li a,b,1, Junpeng Rui b,1, Yuejian Mao a, Anthony Yannarell c, Roderick Mackie a,d,* a Energy Biosciences Institute, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA b Key Laboratory of Environmental and Applied Microbiology, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China c Department of Natural Resources and Environmental Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA d Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA article info abstract

Article history: Rhizosphere bacteria have significant contributions to crop health, productivity and carbon sequestra- Received 24 April 2013 tion. As maize (Zea mays) is an important economic crop, its rhizosphere bacterial communities have Received in revised form been intensively investigated using various approaches. However, low-resolution profiling methods often 27 September 2013 make it difficult to understand the complicated rhizosphere bacterial communities and their dynamics. Accepted 10 October 2013 In this study, we analyzed growth-stage related dynamics of bacterial community structures in the Available online 25 October 2013 rhizosphere of maize using the pyrosequencing method, which revealed an assembly of bacteria enriched in the rhizosphere. Our results revealed that the rhizosphere of maize was preferentially Keywords: Maize rhizosphere colonized by Proteobacteria, Bacteroidetes and Actinobacteria, and each bacterial phylum was repre- Plant growth stage sented by one or two dominating subsets of bacterial groups. Dominant genera enriched in the rhizo- Root exudates sphere included Massilia, Burkholderia, Ralstonia, Dyella, Chitinophaga and Sphingobium. Rhizosphere Bacterial community structure bacterial community structures significantly changed through different growth stages at lower taxo- Pyrosequencing nomic ranks (family, genus and OTU levels). Genera Massilia, Flavobacterium, Arenimonas and Ohtaek- Predominant bacterial genera wangia were relatively abundant at early growth stages, while genera Burkholderia, Ralstonia, Dyella, Dynamics Chitinophaga, Sphingobium, Bradyrhizobium and Variovorax populations were dominant at later stages. Comparisons of pyrosequencing data collected in Illinois, USA in this study with the available data from Braunschweig, Germany indicated many common bacterial inhabitants but also many differences in the structure of bacterial communities, implying that some site-specific factors, such as soil properties, may play important roles in shaping the structure of rhizosphere bacterial community. Ó 2013 Published by Elsevier Ltd.

1. Introduction Singh et al., 2004; Vercellino and Gómez, 2013). Thus, to understand the suite of bacterial interactions that may occur over the The rhizosphere is directly inﬂuenced by root secretions and lifetime of a plant, it is important to know the rhizosphere bacterial associated microorganisms. Soil pH, structure, oxygen and nutrition community and its variation over plant growth stages. levels in the rhizosphere differ from those in the bulk soil (Singh Rhizosphere bacterial communities differ across plant species, et al., 2004). The unique ecological niche shapes the structure of soil type, root architecture and growth stage (Berg and Smalla, rhizosphere bacterial community through the interactions of plant 2009; Marschner et al., 2001, 2004). As maize (Zea mays)isan species, the chemical nature of root exudates, soil properties, and important crop, its rhizosphere bacterial community has been many other factors (Savka and Farrand, 1997). While some detri- intensively investigated using a variety of approaches (Aira et al., mental microbes undermine plant health, mutualistic rhizosphere 2010; Castellanos et al., 2009; Chelius and Triplett, 2001; microbes provide plants with mineral nutrients, phytohormones, Dohrmann et al., 2013). Previous studies suggested that maize se- and protect the plant against phytopathogens (Mendes et al., 2011; lects for a speciﬁc bacterial community depending on soil properties (Castellanos et al., 2009), genotypes (Aira et al., 2010), crop management, such as fertilizer (Aira et al., 2010) and growth stages (Cavaglieri et al., 2009; Di Cello et al., 1997). Some studies have * Corresponding author. 132 Animal Sciences Laboratory, 1207 W. Gregory Drive, suggested that microbial community composition in the maize Urbana, IL 61801, USA. Tel.: þ1 217 2442526; fax: þ1 217 3338286. E-mail address: [email protected] (R. Mackie). rhizosphere is independent of cultivar (Dohrmann et al., 2013; 1 These authors contribute equally. Schmalenberger and Tebbe, 2002), growth stage (Gomes et al.,

0038-0717/$ e see front matter Ó 2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.soilbio.2013.10.017 X. Li et al. / Soil Biology & Biochemistry 68 (2014) 392e401 393

2001) and genotype (Schmalenberger and Tebbe, 2002). The during the growing season (four plots sampled at four time points). apparent disparity of bacterial responses in the rhizosphere could To collect rhizosphere soil, plants were carefully excavated using a be caused partially by differences among plant species and soil shovel, and visible soil particles were removed. Fresh roots were types, but likely also by differences among the methodologies used, collected into a 50 ml centrifuge tube (ca. 50 g). Soil tightly attached such as denaturing gradient gel electrophoresis (DGGE) and clone on the root surface was rinsed using phosphate buffer solution, and library analysis, which have different resolution and detection centrifuged at 8000 rpm for 10 min. The pellet was collected as limitation (Bent and Forney, 2008). Low-resolution profiling rhizosphere soil. Soil cores of 0e30 cm depth between plants were methods potentially miss some important information and make it also collected as bulk soil samples. All the samples were transferred difficult to detail the phylogenetic composition of a rhizosphere to the lab on dry ice, and pre-processed and stored at 80 C for bacterial community. downstream applications. Plant growth stage will influence root physiology, and change the quality and quantity of root exudates; consequently, these 2.2. DNA extraction changes exert a selection on root-associated microorganisms at different growth stages (Dunfield and Germida, 2003; Houlden Soil genomic DNA was extracted using the FastDNA SPIN Kit for et al., 2008). Some studies suggested that seasonal variations in Soil (MP Biomedicals, Solon, OH, USA). DNA quality was assessed by the activity and relative abundance of rhizosphere bacterial com- the ratio of A260/280 and A260/230 using a nanoDrop spectropho- munities are plant-dependent (Dunfield and Germida, 2003; tometer. DNA concentrations were also quantified with Qubit assay Houlden et al., 2008; Mougel et al., 2006; Smalla et al., 2001). (Invitrogen, CA). Although some research showed dynamic changes of maize rhizosphere bacterial community at different growth stages using 2.3. Denaturing gradient gel electrophoresis (DGGE) culture-dependent (Cavaglieri et al., 2009; Nacamulli et al., 1997) and fingerprinting methods (Di Cello et al., 1997), detail phyloge- PCR amplification of bacterial 16S rRNA genes from soil genomic netic compositions cannot be monitored due to low resolution of DNA for DGGE analysis was done using the bacteria-specific for- these approaches. It is also unclear at which taxonomic levels such ward primer 357F containing a GC-clamp and the reverse primer dynamics can be detected. 907R (Muyzer et al., 1993). The PCR mixture (50 ml) contained 1 In this study, we used DGGE and pyrosequencing methods to PCR buffer, 1.5 mM MgCl2, each deoxynucleoside triphosphate at investigate the growth-stage related dynamics of the bacterial 0.4 mM, each primer at 1.0 mM and 1 U of Ex Taq polymerase (TaKaRa composition and diversity in the maize rhizosphere. The objectives Bio Inc, Japan) and 20 ng soil genomic DNA. The PCR amplification are (i) to analyze the bacterial community structure and their program included initial denaturation at 94 C for 3 min, followed growth-stage related dynamics in the rhizosphere of a maize by 30 cycles of 94 C for 40 s, 56 C for 60 s, and 72 C for 60 s, and a cultivar, (ii) to compare the data of this study obtained from a fine- final extension at 72 C for 10 min. PCR products were subjected to silty soil located in Illinois, USA, to those from a similar study electrophoresis using 1.5% agarose gel. The band with a correct size Ò conducted in a silty sand soil in Germany, in order to identify was excised and purified using Wizard SV Gel and PCR Clean-Up common bacterial inhabitants and the difference in the bacterial System (Promega, USA). community structure. DGGE was performed using a DCode system (Bio-Rad, Califor- nia) as described by Muyzer et al. (1993). An 8% polyacrylamide gel 2. Materials and methods with a linear denaturant concentration from 30 to 55% (where 100% denaturant contains 7 M urea and 40% formamide [v:v]) was used 2.1. Field description and sampling to separate the PCR products. Approximately 300 ng of PCR product per lane was loaded onto the DGGE gel. The gel was electro- Our sample site was located at the Energy Farm of the University phoresed for 4 h at 60 C at a constant voltage of 200 V, stained for of Illinois (Champaign, IL, USA, 40030N, 88120W, 230 m elevation). 1 h with SYBR Green I (Molecular Probes, Eugene Org.), illuminated The soil is a Drummer-Flanagan series (fine-silty, mixed, mesic on a transilluminator under UV-light. The sharp bands were Ò Typic Endoaquoll, pH 5.8, organic matter 3.8%, total nitrogen 0.18%) excised. 16S rRNA clone library was constructed using pGEM -T formed from deep deposits of loess and silt parent material on the Easy Vector Systems according to manufacturer’s instruction top of the glacial till and outwash plains. It is an organically rich, (Promega, USA). Ten clones from each sample were sequenced highly productive Corn Belt soil that has been rotation cropped using Sanger method. with maize and legumes for over 100 years. The Z. mays cultivar cv DGGE images were analyzed with Quantity One software (Bio- 34B43 (Pioneer Hi-Bred International, Des Moines, IA, USA) was Rad, USA). A matrix was constructed for all lanes, taking into ac- planted in the middle of May, 2011, in a 0.7-ha field and managed count the presence or absence of the individual bands and the following standard agricultural practices in this region with annual relative contribution of each band to the total intensity of the lane. applications of 20 g m 2 of mixed urea, ammonia, and nitrate fer- A Dice coefficient similarity matrix was generated based on above tilizer at planting. matrix. Finally, DGGE profiles for all samples were clustered based Sampling was conducted after maize emergence at 2 weeks on Dice’s similarity coefficient using the complete-linkage method. (June 1, 2011, vegetative stages V2eV3), 5 weeks (June 21, V8eV9), 9 weeks (July 18, VT, tasseling) and 12 weeks (August 11, R2, blister). 2.4. Pyrosequencing Growth stage was defined based on Hanway’swork(Hanway, 1963). We selected four plots in the maize field for sampling. To amplify the V4-V5 hypervariable regions of 16S rRNA genes for Sampling sites for the next sampling were nearby the previous pyrosequencing, universal primers 519F (50-CAGCMGCCGCGG- locations to minimize the effects caused by spatial variation of soil TAATWC-30) and 926R (50-CCGTCAATTCMTTTRAGTT-30) were used in properties on the bacterial communities. Within each plot, 4 PCR (Baker et al., 2003). The oligonucleotides included 454 Life Sci- rhizosphere soil samples and 4 bulk soil samples were collected ence’s A or B sequencing adapter fused to the 50 end of forward and and composited, so that each individual sample was a pool of four reverse primers. A unique 10-mer barcode sequence was added be- plants or soil cores (1 m away for different plants or soil cores). In tween the sequencing adapter and forward primer to differentiate total, 16 bulk soil and 16 rhizosphere soil samples were obtained between samples. The methods for 16S rRNA gene amplification and 394 X. Li et al. / Soil Biology & Biochemistry 68 (2014) 392e401

PCR product purification for pyrosequencing were the same as those OTUs or bacterial groups with a p-value of 0.05 were selected and described above. Amplicons of all samples were pooled in an equi- identified as the phylotypes or bacterial groups that were signifi- molar library, and then 454 pyrosequencing was carried out using a cantly influenced by rhizosphere or growth stages. GS FLX system (454 Life Sciences, Branford, CT) at Roy J. Carver Biotechnology Center at University of Illinois. 3. Results 2.5. Pyrosequencing data analysis 3.1. DGGE profiles of bacterial communities The raw sequences were sorted based on unique sample tags, DGGE profiling of 16S rRNA fragments from rhizosphere and and trimmed for sequence quality using RDP Pipeline Initial Process bulk soil showed high similarity of DGGE patterns for the four (http://pyro.cme.msu.edu/). Sequences with low quality replicate samples from each sampling time, suggesting a low (length < 300 bp, with ambiguous base ‘N’, and average base variability caused by DGGE sample preparation (field sampling, quality score < 20) were removed, as were any sequences that did DNA extraction, PCR amplification and DGGE analysis). We not perfectly match the proximal PCR primer. The sequences were randomly selected two of four replicates from each sampling time phylogenetically assigned according to their best matches to se- in order to run in one DGGE gel (Fig. 1A). Cluster analysis showed quences in RDP database using the RDP classifier. Sequences an- that DGGE patterns of rhizosphere samples and bulk soil samples notated as chloroplasts or Viridiplantae were removed. The were well separated. In addition, DGGE profiles from early growth processed pyrosequences were aligned using the fast, secondary- stages (June 1 and 21) were clustered together and were distinct structure aware infernal aligner in RDP pyrosequencing pipeline from those of later stages (July 18 and August 11) for both rhizo- (Cole et al., 2009). The aligned 16S rRNA gene sequences were sphere and bulk soil samples. screened for chimeric sequences using the Uchime algorithm The rhizosphere DGGE pattern contained three strong bands (Edgar et al., 2011). Re-sampling to the same sequence depth (2854 that were enriched compared to bulk soil. Band 1 was composed of sequences per sample) was performed using Daisychopper (http:// several bacterial populations, which were not separated by DGGE, www.genomics.ceh.ac.uk/GeneSwytch/Tools.html) prior to down- including phylotypes affiliated to Massilia, Burkholderia and Ral- stream analysis. Sequences were clustered by the complete-linkage stonia. Band 2 contained sequences mainly affiliated to uncultured clustering method incorporated in the RDP pyro pipeline. Opera- Bradyrhizobium (AJ301632, identity 99%), uncultured Lysobacter tional taxonomic units (OTUs) were classified using a 97% nucleo- (AY074793, 98%) and uncultured Steroidobacter (EU979081, 95%). tide sequence similarity cutoff. Rarefaction, Shannon index and Band 3 contained sequences affiliated to uncultured Acidobacteria Chao1 estimator were calculated in RDP at 97% sequence similarity. bacterium (HM062194, 97.5%) and plant chloroplast DNAs. These The phylogenetic affiliation of each 16S rRNA gene sequence was results suggested a strong shift in rhizosphere bacterial community analyzed by RDP Classifier at a confidence level of 80%. across root growth stages. The intensity of band 1 increased at later Dohrmann et al. studied bacterial diversity in the rhizosphere of growth stages, while the intensities of band 2 and band 3 decreased Bt- and conventional maize varieties at the vTI research center in with growth stage. Braunschweig, Germany (521800300N, 102604000E) (Dohrmann et al., 2013). Soil is para brown earth, silty sand (sand 81.8%, silt 8.7%, clay 7.6%, pH 6.4, organic matter 1.67%) (Castellanos et al., 3.2. Overall structural changes of bacterial communities 2009). They measured the bacterial community composition in the samples from different cultivars and root microhabitats (fine or In total, 159,327 high quality and chimera-free reads with an coarse roots). To our knowledge, these datasets are the only average length of 408 bp were obtained (Table SI_1) by pyrose- currently available pyrosequencing datasets of maize rhizosphere quencing. Bacterial diversity measures based on OTU, Chao1 rich- produced using the same PCR primers and DNA extraction method ness, Shannon’s diversity and evenness index revealed the as the current study. Thus, we compared bacterial community enrichment of some phylotypes in rhizosphere compared to bulk composition in these datasets in order to identify the common soil (Table 1). Among 2854 pyrotags, 1058e1234 and 1324e1401 bacterial inhabitants and the differences in bacterial community OTUs (defined at 97% sequence similarity) were found in each structures in maize rhizosphere. The original pyrosequencing data rhizosphere and bulk soil samples, respectively (Table 1, Fig. SI_1). are available at the European Nucleotide Archive by accession PCoA analysis indicated that four randomly collected replicates ERP002580 (current study) and ERP001118 (Germany study) usually clustered closely (Fig. 2), underscoring the reproducibility (http://www.ebi.ac.uk/ena/data/view/). of these bacterial community profiles. The rhizosphere and bulk soil bacterial communities were clearly separated from each other 2.6. Statistical analysis (Fig. 2), indicating a significant impact of maize roots on rhizosphere bacterial community (PerMANOVA, p < 0.05; Table SI_2). Overall structural changes of rhizosphere and bulk soil bacterial Rhizosphere bacterial communities were clearly separated into communities were evaluated by principal coordinates analysis two clusters: an early growth-stage community (first two sampling (PCoA) in Fast UniFrac (http://bmf.colorado.edu/fastunifrac/) points) and a later one (the later two sampling points) (PerMA- (Lozupone et al., 2011). Representative sequences of OTUs were NOVA, p < 0.05) (Fig. 2, Table SI_2), indicating a significant shift of aligned with MUSCLE (Edgar, 2004), and then used to generate a rhizosphere bacterial structure along maize growth stages, even phylogenetic tree using FastTree (Price et al., 2010). The phyloge- though the total number of OTUs and Chao1 did not change netic tree was then used for weighted UniFrac PCoA analysis. The significantly between sampling time points (Table 1). Rhizosphere statistical significance among datasets was assessed by PerMA- effect was the main factor in the first principle coordinate axis NOVA using the weighted PCoA scores in PAST (http://folk.uio.no/ (PCo1), which contributed 46.4% of total variation, and sampling ohammer/past/). A heatmap of the top 150 OTUs selected from the time (growth stage) was the main factor in the second coordinate whole community was constructed using the R package pheatmap axis (PCo2), which contributed 23.1% of variation. The pattern was (http://cran.r-project.org/web/packages/pheatmap/index.html). recapitulated by hierarchical clustering based on UniFrac distance Differences in relative abundances of taxonomic units between matrix (Fig. 2B), which showed similar cluster pattern with DGGE samples were tested by one-way-analysis of variance (ANOVA). profiles. X. Li et al. / Soil Biology & Biochemistry 68 (2014) 392e401 395

Fig. 1. DGGE patterns of PCR amplified 16S rRNA genes from maize rhizosphere and bulk soil (A); clustering of DGGE profiles based on Dice’s similarity coefficient using complete- linkage method (B). Band 1 included phylotypes affiliated to genus Massilia, Burkholderia, Ralstonia; Band 2: Bradyrhizobium, Lysobacter; Band 3: Acidobacteria_Gp1 and chloroplast.

3.3. Taxonomic distributions of bacteria enriched in the rhizosphere 73.2e80.8% of total reads in the rhizosphere and only 46.0e56.2% in of maize bulk soil. As the most abundant phylum, Proteobacteria accounted for 46.2e48.0% of total bacteria in the rhizosphere at early growth The most abundant phyla were Proteobacteria, Acidobacteria, stages, and increased up to 54.7e56.6% at the later growth stages. Actinobacteria, Bacteroidetes and Gemmatimonadetes, accounting On the other hand, the abundance of phyla Acidobacteria, Gem- for 86.4e89.6% and 72.1e73.8% of total bacteria in the rhizosphere matimonadetes, Chloroflexi, Firmicutes and Nitrospira decreased in and bulk soil, respectively. However, the rhizosphere significantly rhizosphere in comparison to bulk soil. These phyla accounted for enriched a subset of phyla (Fig. 3, Table SI_3), which is consistent 25.7e34.3% of total bacteria in bulk soil, but only 11.1e15.7% in the with the lower Shannon diversity of the rhizosphere bacterial rhizosphere. Much higher proportion of unclassified sequences was community (Table 1). Specifically, the rhizosphere enriched Pro- detected in bulk soil, indicating that it contained more unknown teobacteria, Bacteroidetes and Actinobacteria, which accounted for bacterial species.

Table 1 Bacterial diversity indices (at 97% sequence similarity) in the maize rhizosphere and bulk soil.

Soil Jun 1 Jun 21 Jul 18 Aug 11

Number of OTUsa Rhb 1234 20c 1123 33 1058 123 1122 46 Bk 1324 17 1372 46 1401 12 1360 24 Chao1 estimator of richnessd Rh 2817 99 2463 93 2491 271 2448 62 Bk 2807 108 3000 127 3002 75 2928 64 Shannon’s diversity index Rh 6.428 0.053 6.284 0.130 5.947 0.396 6.292 0.106 Bk 6.709 0.039 6.774 0.067 6.856 0.089 6.794 0.049 Shannon’s evenness Rh 0.903 0.006 0.895 0.015 0.854 0.042 0.896 0.010 Bk 0.933 0.004 0.938 0.005 0.946 0.002 0.942 0.005

a All sequence datasets were re-sampled to ensure equal number of reads (2854 reads) for downstream analysis. b Rh: rhizosphere, Bk: bulk soil. c Values are means of four replicate pyrosequencing datasets. d The means of Chao1, Shannon’s diversity and evenness indices were signiﬁcantly different (p < 0.05) between rhizosphere and bulk soil except the samples collected at June 1. All these indices were not signiﬁcantly different between different sampling time in both rhizosphere and bulk soil. 396 X. Li et al. / Soil Biology & Biochemistry 68 (2014) 392e401

Fig. 2. Overall structural changes of maize rhizosphere (Rh) and bulk soil (Bk) bacterial communities from pyrosequencing data. (A) PCoA score plot based on weighted UniFrac metrics; (B) clustering of soil bacterial communities based on the UniFrac distances calculated in Fast UniFrac. Early stage samples were collected at June 1 and June 21 (sample A and B in ﬁgure B); later stage samples were collected at July 18 and August 11 (sample C and D in ﬁgure B). Detail sample information was shown in Table SI-1.

The most dominant classes in the rhizosphere were Betapro- These OTUs included OTU27365 (Chitinophaga), OTU5987 (Strep- teobacteria, Actinobacteria, Alphaproteobacteria, Gammaproteo- tomyces), OTU5729 (Sphingobium), OTU3354 (Sphingobium) and bacteria and Sphingobacteria, which accounted for 63.1e67.4% of OTU1352 (Sphingobium). total bacteria at the early stages and 76.4e77.2% at the later stages (Table SI_3, Fig. SI_2). Within the rhizosphere Proteobacterial 3.4. Growth-stage related dynamics of the bacterial community community at early growth stages, there was over-representation structure of Betaproteobacteria families compared to Alphaproteobacteria and Gammaproteobacteria. However, the relative proportion of The structures of early growth-stage communities were signifi- Alphaproteobacteria was the highest in August samples. cant different from those of later growth stages in both rhizosphere Lower-order taxonomic analysis demonstrated that a specific and bulk soil (Fig. 2 and Table SI_2). At the phylum level, the relative subset of Proteobacteria was enriched in the rhizosphere, including abundance of Proteobacteria and Actinobacteria increased signifi- Xanthomonadaceae (mainly including genera Dyella and Arenimo- cantly with growth stage in both rhizosphere and bulk soil nas), Oxalobacteraceae (mainly genus Massilia), Burkholderiaceae (Table SI_3), while the relative abundance of Acidobacteria, Bacter- (mainly genera Burkholderia and Ralstonia) and Sphingomonadaceae oidetes and Gemmatimonadetes decreased. Moreover, at the phylum, (genus Sphingobium)(Fig. 3). The family Streptomycetaceae (genus class and order levels, the dynamic patterns of many bacterial com- Streptomyces) in Actinobacteria, and the families Chitinophagaceae munities were comparable in rhizosphere and bulk soil (Fig. SI_4). (genus Chitinophaga) and Flavobacteriaceae (genus Flavobacterium) At the family level, Oxalobacteraceae decreased at later stages in in Bacteroidetes were also enriched in the rhizosphere. rhizosphere soil, while Burkholderiaceae and Sphingomonadaceae At the genus level, 333 genera were detected in the rhizosphere. increased significantly (p < 0.05) (Fig. 4). Members of these families Eighty-nine genera were identified as significantly enriched in the did not change significantly in the bulk soil (Table 2). At the genus rhizosphere (p < 0.05) compared to bulk soil (Table SI_3). Among level, Massilia, Flavobacterium and Arenimonas dominated in the them, 49 genera were significantly enriched at early growth stages, early growth-stage bacterial community; Ralstonia, Burkholderia, and 67 genera were significantly enriched at the later stages. Ohtaekwangia and Bradyrhizobium dominated in the middle growth Twenty-two genera were identified to be dominant genera with period; Chitinophaga, Dyella, Sphingobium and Variovorax domi- relative abundance > 0.5% of total bacteria reads in the rhizosphere, nated in the later growth stage (Fig. SI_5). Massilia was the most and these genera occurred in all the rhizosphere and most bulk soil abundant genus in the early growth stages, and it sharply decreased samples (Table 2). Among them, 18 enriched genera were repre- in the late stages. The changes in Massilia proportion were mainly sented with relative abundance > 1% in the rhizosphere, and driven by the shifts in three dominant OTUs (OTU6022, 5806, together they accounted for average 33.5% of total reads in the 3337). However, these genera did not change significantly in the rhizosphere and 3.5% in the bulk soil (Fig. SI_3). These most bulk soil. In bulk soil, genera Gp4 (Acidobacteria), Nitrospira, Pas- abundant genera mainly included Massilia, Burkholderia, Chitino- teuria, Ohtaekwangia and Arenimonas decreased significantly at the phaga, Ralstonia, Dyella, Sphingobium and Flavobacterium. later stages, while genera Ktedonobacter and Bradyrhizobium At the OTU level, seventy-five OTUs were identified as signifi- increased at the later stages (Table SI_3, Fig. SI_6). cantly enriched in the rhizosphere (Table SI_3). Among them, 28 OTUs were identified as dominant enriched OTUs with relative 3.5. Comparisons of Illinois datasets with German datasets abundance > 0.5% in at least one sample (Table 2). The most abundant 28 rhizosphere OTUs accounted for 22.5% of total reads PCoA analysis indicated that bacterial communities from on average. These OTUs were mainly affiliated with the same German maize rhizosphere clustered together regardless of culti- abundant genera mentioned above. Among 28 OTUs, 5 OTUs were vars or root microhabitats (fine or coarse roots), and they were very low or not detected in some early stage samples, but they significantly different from Illinois energy farm samples (p < 0.01 appeared in the late stage samples with relative high abundance. by PerMANOVA) (Fig. SI_7). The differences between German and X. Li et al. / Soil Biology & Biochemistry 68 (2014) 392e401 397

Fig. 3. Phylogenetic distributions of phyla (% of total reads) present in the rhizosphere and bulk soil (A), and relative abundance (% of individual taxonomic group) of classiﬁed families in the phyla Actinobacteria (B), Bacteroidetes (C) and Proteobacteria (D) and in class Alphaproteobacteria (E), Betaproteobacteria (F) and Gammaproteobacteria (G). Dis- tribution of classiﬁed genera in two dominant Proteobacteria orders of Burkholderiales (H) and Xanthomonadales (I).

Illinois rhizosphere samples were larger than the differences (genus Massilia, enriched at the early stage) and Burkholderiaceae among the maize cultivars, root microhabitats or growth stages. (genera Burkholderia and Ralstonia, enriched at the late stage), than At high taxonomic ranks, all the rhizosphere samples enriched German samples. In addition, Illinois samples also contained more Proteobacteria and Actinobacteria. However, Illinois samples con- Xanthomonadaceae (genus Dyella) and Chitinophagaceae (genus tained more Bacteroidetes and less Firmicutes. When we compared Chitinophaga), but less Nocardioidaceae, Pseudomonadaceae these datasets at lower taxonomic ranks, different patterns were (genus Rhizobacter) and genus Arthrobacter. observed (Table SI_4). Illinois samples contained more bacteria In total 34,456 OTUs were detected in the combining dataset belonging to order Burkholderiales, such as Oxalobacteraceae that contained 518,576 pyrosequencing reads from both Illinois and 398 X. Li et al. / Soil Biology & Biochemistry 68 (2014) 392e401

Table 2 Dominant genera and OTUs signiﬁcantly enriched or depleted (p < 0.05) in the rhizosphere with relative abundance > 0.5% of total bacterial reads.

Time Relative abundance in Rh (%) Relative abundance in Bk (%)

Jun 1 Jun 21 Jul 18 Aug 11 Jun 1 Jun 21 Jul 18 Aug 11

(a) Enriched genera (22) Ralstonia 0.096 0.858 9.294 1.384 0.009 0.000 0.018 0.000 Sphingobium 0.044c 0.184c 4.143b 6.430a 0.018 0.018 0.009 0.044 Duganella 0.710b 1.831a 0.832b 0.946b 0.018 0.018 0.018 0.088 Massilia 11.361a 10.363a 1.752b 2.094b 0.324a 0.254ab 0.070b 0.377a Variovorax 0.482c 1.051bc 1.314ab 1.901a 0.070 0.018 0.070 0.044 Chitinophaga 0.815b 2.461ab 3.968ab 5.019a 0.149ab 0.184ab 0.061b 0.140ab Burkholderia 1.384b 1.585b 5.694a 5.168a 0.035c 0.096bc 0.175ab 0.298a Stenotrophomonas 0.420 0.149 0.701 1.148 0.105 0.000 0.000 0.009 Mucilaginibacter 1.585a 1.612a 0.876ab 0.491b 0.070ab 0.114a 0.000b 0.044ab Niastella 0.841b 2.356a 0.596b 0.578b 0.018c 0.070bc 0.175a 0.123ab Pseudomonas 1.463a 0.333b 0.315b 0.333b 0.044 0.053 0.026 0.096 Rhizobium 0.219b 0.307b 1.515a 0.999a 0.044b 0.053b 0.035b 0.158a Dyella 1.594 2.120 3.618 3.723 0.193 0.491 0.149 0.736 Streptomyces 1.016b 2.356a 1.384b 1.156b 0.175b 0.184bc 0.254b 0.543a Flavobacterium 3.136a 1.454ab 0.745b 0.368b 0.797a 0.324b 0.079b 0.114b Lysobacter 0.237b 0.762a 0.473ab 0.631ab 0.175a 0.131ab 0.061b 0.140ab Sphingomonas 0.272b 0.315b 0.578b 0.893a 0.053b 0.061b 0.035b 0.420a Arenimonas 1.892a 1.840a 0.526b 0.806b 0.972a 0.631ab 0.368b 0.368b Ohtaekwangia 1.244a 1.515a 0.377b 0.350b 0.543a 0.657a 0.184b 0.298b Marmoricola 0.438 0.569 0.499 0.806 0.175b 0.175b 0.350b 0.622a Bradyrhizobium 0.158b 0.359b 1.200a 1.148a 0.149b 0.359b 0.841a 0.745a Nocardioides 0.788 0.552 0.797 0.797 0.298b 0.394b 0.491b 1.244a

(b) Depleted genera Acidobacteria Gp1 2.786a 2.032ab 1.472b 1.883ab 3.066ab 3.013ab 3.416a 1.761b Rhizomicrobium 0.674ab 0.368b 0.350b 1.034a 1.174a 0.850ab 0.648b 0.745ab Terrimonas 0.359 0.447 0.272 0.245 0.771a 0.561ab 0.674ab 0.385b Gemmatimonas 2.488a 2.225a 1.130b 1.358b 3.986a 3.784a 3.285a 2.330b Acidobacteria Gp7 0.596a 0.280b 0.298b 0.403ab 0.797 0.736 0.788 0.745 Acidobacteria Gp3 0.578 0.552 0.946 0.999 1.296b 1.209b 2.374a 1.621b Acidobacteria Gp6 1.638a 1.323ab 0.850b 0.780b 2.645 3.022 3.127 2.199 Acidobacteria Gp10 0.228 0.193 0.105 0.114 0.482 0.438 0.596 0.508 Pasteuria 0.298a 0.263a 0.158ab 0.088b 0.893a 1.060a 0.456b 0.429b Nitrospira 0.911a 0.657ab 0.683ab 0.403b 3.591a 2.882b 2.225b 1.402c Chthonomonas 0.307a 0.140b 0.280ab 0.193ab 1.156a 0.823ab 0.955ab 0.587b Acidobacteria Gp4 2.672a 2.628a 1.165b 1.349b 12.158a 8.847bc 7.665bc 5.019c Ktedonobacter 0.026c 0.123bc 0.193ab 0.254a 0.201b 0.412b 0.727b 3.294a

(c) Enriched OTUs (28) OTU27365 (Chitinophaga) 0.000 0.044 1.375 0.000 0.000 0.000 0.000 0.000 OTU5987 (Streptomyces) 0.000b 0.210ab 0.701a 0.350ab 0.000 0.000 0.000 0.000 OTU5543 (Ralstonia) 0.070 0.657 6.473 0.920 0.000 0.000 0.018 0.000 OTU5996 (Ralstonia) 0.026 0.201 2.742 0.438 0.009 0.000 0.000 0.000 OTU5729 (Sphingobium) 0.000b 0.026b 1.156a 0.990a 0.000 0.000 0.000 0.009 OTU6022 (Massilia) 5.764a 5.808a 0.639b 0.403b 0.009 0.009 0.009 0.026 OTU3354 (Sphingobium) 0.000c 0.070c 1.656b 2.593a 0.000 0.009 0.009 0.009 OTU5787 (Dyella) 0.044b 0.508ab 1.410a 0.841ab 0.009 0.009 0.000 0.000 OTU3337 (Massilia) 1.866a 1.148b 0.403bc 0.710c 0.009 0.000 0.000 0.018 OTU1352 (Sphingobium) 0.009c 0.053c 1.218b 2.689a 0.000 0.009 0.000 0.018 OTU5682 (Burkholderia) 0.359 0.482 1.349 1.086 0.000 0.000 0.009 0.026 OTU5806 (Massilia) 2.645a 2.680a 0.447b 0.622b 0.000 0.026 0.000 0.044 OTU6053 (Dyella) 0.044b 0.272b 0.385b 0.745a 0.000 0.000 0.018 0.000 OTU5568 (Variovorax) 0.070b 0.131b 0.412b 0.780a 0.009 0.000 0.000 0.009 OTU2967 (Chitinophaga) 0.018b 0.166ab 0.438ab 0.674a 0.009 0.000 0.000 0.009 OTU3498 (Unclassiﬁed Enterobacteriaceae) 0.517ab 0.044b 1.629a 0.333b 0.000 0.009 0.018 0.035 OTU5591 (Burkholderia) 0.096b 0.158b 0.972a 0.464ab 0.000 0.000 0.009 0.035 OTU1497 (Dyella) 0.718 0.797 1.612 1.918 0.035 0.070 0.009 0.026 OTU348 (Duganella) 0.534b 1.568a 0.613b 0.788b 0.000b 0.018b 0.018b 0.070a OTU519 (Burkholderia) 0.315b 0.385b 1.743a 1.691a 0.018 0.053 0.044 0.035 OTU3364 (Chitinophaga) 0.070c 0.613b 0.298bc 1.375a 0.009 0.018 0.026 0.044 OTU5214 (Rhizobium) 0.105b 0.096b 0.780a 0.508a 0.000b 0.009b 0.009b 0.061a OTU5889 (Niastella) 0.298b 0.937a 0.140b 0.158b 0.009b 0.000b 0.061a 0.026ab OTU3131 (Streptomyces) 0.666 0.858 0.526 0.683 0.018 0.018 0.061 0.088 OTU165 (Arenimonas) 0.841a 0.631a 0.123b 0.333b 0.307 0.201 0.149 0.140 OTU200 (Unclassiﬁed Burkholderiales incertae sedis) 0.456ab 0.631a 0.193b 0.219b 0.307a 0.088b 0.088b 0.140b OTU875 (Arenimonas) 0.499a 0.578a 0.184b 0.149b 0.324a 0.210ab 0.114b 0.079b OTU242 (Bradyrhizobium) 0.114b 0.254b 0.841a 0.815a 0.123c 0.228bc 0.526a 0.447ab

(d) Depleted OTUs OTU82 (Acidobacteria Gp4) 0.420a 0.403a 0.123b 0.140b 1.437a 1.226a 1.007ab 0.674b OTU11 (Acidobacteria Gp4) 0.447a 0.272ab 0.158bc 0.061c 1.463a 1.340ab 1.165ab 0.736b X. Li et al. / Soil Biology & Biochemistry 68 (2014) 392e401 399

Table 2 (continued )

Time Relative abundance in Rh (%) Relative abundance in Bk (%)

Jun 1 Jun 21 Jul 18 Aug 11 Jun 1 Jun 21 Jul 18 Aug 11

OTU189 (Unclassiﬁed Chitinophagaceae) 0.131 0.096 0.044 0.149 0.692a 0.780a 0.429ab 0.263b OTU26 (Acidobacteria Gp4) 0.166b 0.464a 0.061b 0.105b 1.638a 1.445ab 0.692bc 0.359c OTU230 (Nitrospira) 0.140 0.114 0.053 0.044 0.771a 0.964a 0.272b 0.298b

Values labeled with different letters in a row indicated signiﬁcant at p < 0.05.

German rhizosphere samples. 4800 OTUs (13.9% of total numbers of of the plant (Fig. 4). Early growth-stage related communities were OTUs) were shared in both datasets, and they accounted for 70.3% clearly separated from later ones (Fig. 2). Overall, members of phyla of total reads in Illinois samples and 68.6% in German samples, Proteobacteria (class Betaproteobacteria and Gammaproteobac- indicating that most root-associated bacteria were shared in both teria) and Bacteroidetes (class Sphingobacteria) were the most sites. In this combined dataset, the average relative abundance of 14 enriched bacteria in maize rhizosphere (Fig. 3). This is considered to OTUs in Illinois samples and 11 OTUs in German samples were be a carbon-rich environment, and ecological theory suggests that higher than 0.5% of total reads (Table SI_4). These OTUs were copiotrophic behavior or r-selected populations should be well mainly affiliated to genera Massilia, Ralstonia, Sphingobium, Bur- adapted to such nutrient-rich conditions. Many Betaproteobacteria kholderia and Duganella in Illinois samples, while Massilia, Arthro- and Bacteroidetes are copiotrophic soil bacteria, and they become bacter, Rhizobacter and Streptomyces in German samples. Four OTUs abundant when labile substrates are available (Fierer et al., 2007; occurred only in Illinois samples, including OTU3070b (Bur- Goldfarb et al., 2011). Our results confirmed that these organisms kholderia), 30638b (Ralstonia), 2068b (Dyella) and 6727b (Dyella). predominate in the rhizosphere, presumably because labile organic Though most of the dominant genera were shared in both Illinois substrates are more available than in bulk soil. In addition, Bur- and German samples, important OTUs in the same genus were kholderiaceae, Xanthomonadales and Actinobacteria harbor genera quite different, for example, in the genus Massilia, OTU60b was and species with anti-microbial activities (Mendes et al., 2011), more abundant in German samples, while five other OTUs which may enable them to operate as interference competitors in (OTU1234b, 2383b, 298b, 4839b and 3722b) were more abundant the rhizosphere. in Illinois samples. The DGGE profiles in this study revealed a shift in a few dominant bacterial populations over the course of maize growth (Fig. 1). 4. Discussion Because DGGE showed similar dynamic patterns as those revealed by pyrosequencing, it is a useful technique to give a snapshot of the We detected the dominant bacterial taxa enriched in the maize diversity of the most abundant rhizosphere bacteria (Gomes et al., rhizosphere using the high-resolution pyrosequencing technique 2001; Smalla et al., 2001). However, pyrosequencing results pro- (Table 2), and we found that changes in the maize rhizosphere vided more specific insight into shifts in bacteria community community composition were strongly linked to the growth stage composition at the levels of family, genus and OTU (Fig. 4). Dynamic

Fig. 4. Growth-stage related dynamic changes in relative abundances (% to total reads) of dominant bacterial groups at family, genus and OTU levels. 400 X. Li et al. / Soil Biology & Biochemistry 68 (2014) 392e401 changes of many bacterial groups in the rhizosphere were similar to In addition to changes in rhizosphere community composition those in bulk soil at high taxonomic ranks; for example class over time, we also documented variability that may be attributable Actinobacteria (mainly order Actinomycetales) and order Rhizo- to environmental conditions. The comparison of our results, ob- biales (Fig. SI_2) both displayed similar temporal shifts in the tained from a fine-silty soil in Illinois, USA, to those from a silty sand rhizosphere and bulk soil. It may be that seasonal climate change soil in Germany (Dohrmann et al., 2013) showed significantly impacts these bacteria in both rhizosphere and bulk soils. different bacterial communities at the level of family and below However, there were several key microbial groups (at the level (Table SI_4). The number of reads belonging to shared OTUs of family and below) that displayed different temporal dynamics in accounted for about 70% of total bacterial reads in samples from the rhizosphere from that in the bulk soil. In fact, many of the both sites, and this suggests that the maize rhizosphere generally dominant rhizosphere genera (Massilia, Chitinophaga, Sphingobium, selects for similar dominant members independent of variety, age etc.) displayed different patterns of temporal changes in the or soil type. However, the number of shared OTUs only accounted rhizosphere as opposed to the bulk soil (Fig. 4), and we propose that for 13.9% of total observed OTUs, and this implies that most of site- the dynamics of these rhizosphere bacteria were largely plant- specific OTUs are rare species. The differences in bacterial com- driven and not due to direct seasonal effects. It can be expected munity structure of maize rhizosphere between Illinois and Ger- that the composition and chemical nature of root exudates will many were larger than the differences between cultivars (German change over the plant growth cycle (Dunfield and Germida, 2003), samples), root microhabitats (German samples) or growth stages and these changes should alter the nutritional environment of (Illinois; Fig. SI_7). Another study conducted in the carbonate-rich rhizosphere bacteria. Bacteria present in the rhizosphere of young soil (pH 8.5) using 16S rRNA clone library analysis showed that plants preferentially utilize simple amino acids, whereas bacteria Pseudomonas and Lysobacter genera constituted 45% of the total from mature plants utilize more complex carbohydrates (Houlden bacterial abundance in the maize rhizosphere (García-Salamanca et al., 2008). It is also recognized that copiotrophic behavior or r- et al., 2013). Though exact mechanisms for such differences are selected populations are positively selected in early stages of mi- not completely understood, because these studies were all con- crobial succession, while oligotrophic or k-selected populations are ducted on different soil types, it is likely that soil type may be one of positively selected in later stages (Fierer et al., 2007). Our results the most important environmental variables influencing the support the conclusion that dominant bacteria species in the early structure of the maize root-inhabiting bacterial community at low growth-stage communities are characteristic of fast-growing, taxonomic levels (Berg and Smalla, 2009; Castellanos et al., 2009; copiotrophic bacteria that are competent at utilizing labile Garbeva et al., 2004). Growth-stage related dynamics and the dif- organic substrates. ferences in rhizosphere bacterial community structure from In this study, we found that significant growth-related dy- different soil types further support the general concept that plant namic changes in bacterial community structure were mainly and soil type cooperatively shape the structure of microbial com- associated with phylum Proteobacteria or Bacteroidetes, espe- munity in the rhizosphere (Berg and Smalla, 2009). cially order Burkholderiales (mainly genera Massilia, Burkholderia In conclusion, the pyrosequencing surveys in this study and Variovorax)(Fig. 4 and Fig. SI_2). Members of Burkholderiales revealed, in detail, the composition and dynamics of bacterial were enriched in the rhizosphere, possibly due to their versatile community structure in the maize rhizosphere. These communities abilities to utilize root metabolites, degrade aromatic compounds are dominated by members of the genera Massilia, Burkholderia, (Goldfarb et al., 2011; Vandenkoornhuyse et al., 2007) and pro- Ralstonia, Dyella, Chitinophaga and Sphingobium. Many of these duce anti-microbial substances (Coenye and Vandamme, 2003). In rhizosphere bacteria, especially order Burkholderiales, showed this order, genera Massilia predominated at the early stages of active, dynamic changes with root growth stage, and there appears succession, while Burkholderia and Variovorax increased at later to be, in general, an early growth stage community that is distinct growth stages (Fig. 4, Fig. SI_5). In addition to Massilia, the genera from those of later stages. The comparisons of bacterial community Flavobacterium, Arenimonas and Ohtaekwangia also appeared at structures from Illinois soil and Germany soil supported the notion early stages of succession, indicating that they may be copio- that soil types may be very important environmental variables trophic and fast-growth bacteria, able to exploit a transient niches influencing the structure of the maize root-inhabiting bacterial for nutrition and others at the early growth stage. Ofek et al. also community. Future investigation is warranted into whether the reported that the dominance of Massilia may be attributed to its pattern of growth-related dynamics in rhizosphere bacteria com- high proliferation rates when sufficient carbon and energy sour- munity is specific with particular cultivars, root segments and/or ces are present (Ofek et al., 2012). The decline of Massilia at later soil types. stages may be explained by the reduction of quantity and quality of organic carbon, or interspecies competition for resources. The Acknowledgments relative abundance of genera Chitinophaga and Sphingobium sharply increased at later stages in similar proportion to the This work was funded by the Energy Biosciences Institute, genus Burkholderia. Members of genus Chitinophaga are chitino- Environmental Impact and Sustainability of Feedstock Production lytic (Kim and Jung, 2007), while Sphingobium and Burkholderia Program at the University of Illinois, Urbana. We thank Dr. Benli can typically degrade lignin-derived aromatic compounds Chai for the help in data treatment. (Takeuchi et al., 2001; Vandenkoornhuyse et al., 2007). The ability of Burkholderia, Chitinophaga and Sphingobium to use polymers Appendix A. Supplementary data may enable them to be more competitive for colonization of mature roots. Supplementary data related to this article can be found at http:// Although bulk soil is recognized to be highly stable in commu- dx.doi.org/10.1016/j.soilbio.2013.10.017. nity structure and function (Houlden et al., 2008), our pyrosequencing results also revealed significant seasonal changes of some soil genera and OTUs (Fig. 4, Fig. SI_6). For example, the relative References abundance of genera Gp4 (Acidobacteria), Nitrospira, Pasteuria, Aira, M., Gomez-Brandon, M., Lazcano, C., Baath, E., Dominguez, J., 2010. Plant ge- Ohtaekwangia and Arenimonas decreased late in the growth season, notype strongly modifies the structure and growth of maize rhizosphere mi- while Ktedonobacter and Bradyrhizobium increased. crobial communities. Soil Biol. Biochem. 42, 2276e2281. X. Li et al. / Soil Biology & Biochemistry 68 (2014) 392e401 401

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