Journal of Integrative Agriculture 2016, 15(8): 1883–1891

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RESEARCH ARTICLE

Characterization of root-associated bacterial community structures in soybean and corn using locked nucleic acid (LNA) oligonucleotide- PCR clamping and 454 pyrosequencing

YU Zhen-hua1*, YU Jiang1, 2*, Makoto Ikenaga3, Masao Sakai3, LIU Xiao-bing1, WANG Guang-hua1

1 Key Laboratory of Mollisols Agroecology, Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Harbin 150081, P.R.China 2 College of Agronomy, Northeast Agricultural University, Harbin 150030, P.R.China 3 Faculty of Agriculture, Kagoshima University, Kagoshima 890-0065, Japan

Abstract The community structure and diversity of root-associated have been tentatively investigated using polymerase chain reaction (PCR) amplification methods in several studies. However, the homology between small submit ribosomal (SSU) rRNA genes of plant plastids and mitochondria and that of bacteria have hindered in these studies. To address this issue, in this paper, we adopted the methods of locked nucleic acid (LNA) oligonucleotide-PCR clamping with 454 pyrosequencing to analysis the root-associated bacterial community compositions in soybean and corn. Results showed that plant chloroplast and mitochondria genes were effectively inhibited from PCR amplification in the root samples with LNA oligonucleotides (LNA (+)), and PCR amplicons with LNA (+) had higher bacterial operational taxonomic unit (OTU) numbers and ACE, Chao1, and Shannon indices, as well as a lower Simpson index than the corresponding samples with- out LNA oligonucleotides (LNA (–)). Those findings suggested that the methods of this study provide a much more detail description of root-associated bacterial communities. In the soybean LNA (+) sample, Pseudomonas, Bradyrhizobium and Flavobacterium were the three most abundant genera, whereas the top two predominant genera in corn LNA (+) samples were Streptomyces and . The presence and absence of major genera varied between soybean and corn, suggesting the root-associated bacterial communities differed between two crops. The rare phylotypes and uncultured root-associated bacterial members detected in this study inferred that the root-associated bacterial communities are highly complex and information on their taxonomic affiliates potentially gives the clues for selecting the optimal medium and method to isolate the novel bacteria for further functional analysis.

Keywords: bacterial diversity, high-throughput sequencing, corn, soybean, SSU rRNA genes

Received 26 June, 2015 Accepted 12 October, 2015 Corresponddence WANG Guang-hua, Tel: +86-451-86602745, Fax: +86-451-86603736, E-mail: [email protected]; Makoto 1. Introduction Ikenaga, Tel: +81-99-2858660, E-mail: [email protected]. ac.jp * Plant-associated bacteria, including rhizobacteria, epiphytes These authors contributed equally to this study. and endophytes, have been isolated from a variety of plant © 2016, CAAS. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http:// tissues (Mano et al. 2006; Ferrando et al. 2012). Extensive creativecommons.org/licenses/by-nc-nd/4.0/) studies have shown that these bacteria exert beneficial ef- doi: 10.1016/S2095-3119(15)61195-9 fects on their host plants by establishing resistance to heavy 1884 YU Zhen-hua et al. Journal of Integrative Agriculture 2016, 15(8): 1883–1891

metals, producing plant hormones, enhancing the fixation emerged as a powerful platform for DNA analysis (Ronaghi of nitrogen, protecting their host plants against pathogen 2001). This method can generate one million base pair (bp) infection, or increasing the uptake of mineral nutrition reads and can provide a much more detail description of (Bashan et al. 2004; Sessitsch et al. 2004; Germaine et al. the microbial communities than traditional methods such as 2006). Therefore, a better understanding of the functions cloning library, DGGE, and PLFA. Since this technique was and potential roles of plant-associated bacteria is critical invented, it has been shown to be a very powerful technique for keeping plant health. Plant roots are the main habitats in investigating microbial communities in various environ- of indigenous endophytic bacteria with larger population ments (Acosta-Martinez et al. 2008; Peay et al. 2013) but number than other plant tissues (Hallmann et al. 1997). limited to root-associated microbes. In this study, we com- Therefore, the roots are the most important plant tissues bined using the LNA-PCR clamping and 454 pyrosequenc- for investigating the diversity and composition of plant-as- ing methods in investigating the diversity and composition of sociated bacterial communities in various plants (Gardner root-associated bacterial communities of soybean and corn. et al. 1982; Li et al. 2011). The purpose of this study were (1) to further demonstrate Comprehensive characterization of plant-associated the advantage of using the LNA technique in investigating bacterial communities had been limited by a number of plant-associated bacterial communities, (2) to characterize methodological constraints. First, majority of plant-associ- the diversity and composition of root-associated bacteria of ated bacteria like those in the soils are non-culturable and soybean and corn, and (3) to identify the dominant and novel can only be detected by culture-independent techniques members of root-associated bacteria between two crops. (Tholozan et al. 1999; Mazzaglia et al. 2001). Second, the culture-independent method of PCR amplification of 16S 2. Materials and methods rDNA sequences is restricted to investigate the compositions of plant-associated bacteria, since the small submit ribosom- 2.1. Study site and preparation of root samples al (SSU) rRNA genes of plant plastids and mitochondria are highly homologous to that of bacterial rRNA genes, which Root samples of soybean (Glycine max cv. Dongsheng 1) resulting in high proportion of plant organelle sequences and corn (Zea mays cv. Xinken 5) were collected from an was detected in the PCR amplicons (Lane 1991; Rastogi experimental field in Guangrong Village, Hailun City, Hei- et al. 2010). longjiang Province of Northeast China (47°21´N,126°49´E), To reduce DNA contamination of plant organelles, Che- which was established in the fall of 2004. The experimen- lius and Triplett (2001) designed primer 799f with 1492r to tal site has a mean annual temperature of 1.5°C, and a PCR amplify bacterial sequences directly from corn roots, mean annual precipitation of approximately 500–600 mm, and this primer set has been extensively employed in the with 70% occurring in July, August and September. The study of plant endophytic bacterial community (Sun et al. common cropping system of the field was soybean-corn 2008). Although primer 799f was highly specific to most rotation. The soil is the typical Mollisol with the content of bacterial SSU rRNA gene sequences, mitochondrial rRNA total organic carbon and total nitrogen was 31.4 and 1.81 g gene sequences are often detected in the PCR products kg–1, respectively. Roots samples were collected at the (Chelius and Triplett 2001). To overcome these drawbacks, flowering stage of soybean and 3-leaf stage of corn under Ikenaga and Sakai (2014) developed the locked nucleic acid chemical fertilizer-treated plots. Three plant roots were (LNA) technique to specific amplify the SSU rRNA genes collected and pooled together in a single polyethylene bag, of bacteria. Results showed that using locked nucleic acid then transported to the laboratory at 4°C. The roots were (LNA) oligonucleotides selectively amplified bacterial genes then immediately washed several times with tap water to while inhibited the amplification of organelle genes derived remove adhering soil particles and then completely sub- from rice, corn, soybean, and potato (Ikenaga and Sakai merged in 0.05% sterilized sodium dodecyl sulfate (SDS) 2014; Ikenaga et al. 2015). Therefore, this technique was solution with hand shaking for about 10 times. The SDS considered as one of effective ways to study the community solution was then discarded, and the roots were washed structure of plant-associated bacteria. In their pioneering several times again in sterilized water until no SDS foam works, the denaturing gradient gel electrophoresis (DGGE) was left. Subsequently, the roots were cut into about 2-mm method was subjected to imaging the root-associated bac- size portions with sterilized scissors and then ground with a terial community (Ikenaga and Sakai 2014; Ikenaga et al. sterilized mortar and pestle for DNA extraction. 2015), however, a few DGGE bands were observed in the gel which interferes complete description of root-associated 2.2. DNA extraction from root samples bacterial communities. Recently, bar-coded 454 pyrosequencing has been The DNA was extracted from the triplicate ground root YU Zhen-hua et al. Journal of Integrative Agriculture 2016, 15(8): 1883–1891 1885

samples (about 0.5 g fresh weight) using Fast DNA SPIN Kit Technology Co., Ltd., Shanghai, China. for Soil (MP Biomedicals, Solon, OH, USA), based on bead beating following the manufacturer’s instructions. Extracted 2.4. Processing of pyrosequencing data and diversity DNA was stored at –20°C until use. indices

2.3. PCR amplification and bar-coded pyrosequencing After sequencing was completed, all sequence reads were quality checked using the quantitative insights into micro- An aliquot of the extracted DNA from each sample was used bial ecology (QIIME) pipeline (ver. 1.17; http://qiime.org/). as template for the first run of nested PCR amplification Any ambiguous reads were excluded from further analysis. based on the previous reporters (Ikenaga and Sakai 2014; Bacterial sequences with the same barcode were assigned Ikenaga et al. 2015). The sequences of LNA oligonucle- into the same sample, and then the barcode and primer otides which used to inhibit the amplification of corn and sequences were removed. Sequences with similarities of soybean plastid and mitochondria SSU rRNA genes, and >97% were clustered into one operational taxonomic unit bacterial primers 63f-mi/1492r-y are shown in Appendix A. (OTU). Rarefaction curves, coverage, and diversity indices A DNA extraction from soybean and corn roots was also (ACE, Chao1, Shannon, and Simpson) were obtained using directly amplified with bacterial primers 63f-mi/1492r-y the MOTHUR program (http://www.mothur.org). Phylotypes without using LNA oligonucleotides as control, respec- were identified using Ribosomal Database Project (RDP) tively. The PCR mixture contained 12.5 μL Premix Hot pyrosequencing pipeline (http:// pyro.cme.msu.edu/). All Start Version (TaKaRa, Otsu, Japan), 1.0 μL primer 63f-mi sequences have been deposited into the GenBank short- (20 pmol μL–1), 1.0 μL of primer1492r-y (20 pmol μL–1), 3.75 μL read archive SRP051225. of LNA-Mit63 (20 pmol μL–1), 3.75 μL of LNA-Mit1492 (20 pmol μL–1), 1.25 μL LNA-Pla63a/b (10 pmol μL–1), and 3. Results 1.25 μL LNA-Pla 1492a/b (10 pmol μL–1). A 0.5-μL aliquot of the DNA template was added to a total volume of 25 μL. 3.1. Comparison of PCR products with or without The amplification condition was as follows: 94°C for 3 min LNA oligonucleotides (initial denaturation), followed by 30 cycles of 94°C for 1 min, 70°C for 1 min (annealing step of LNA oligonucleotides), According to the PCR amplification results of Ikenaga and 54°C for 1 min (annealing step of primers), and 72°C for Sakai (2014), the PCR profile of the amplified mitochondria 2 min, with the final extension step at 72°C for 10 min. PCR and plastid genes without LNA oligonucleotides was similar to products were visualized under ultraviolet (UV) light on a that of aseptic roots which generate mitochondria and plastid 1.5% agarose gel stained with ethidium bromide to ensure products only, and the size of the amplified bacterial genes the expected size for the targeted sequence. The PCR was between that of the mitochondria and plastid genes. products were then purified with High Pure PCR Product In this study, the first round of PCR products of SSU rRNA Purification Kit (Roche, Indianapolis IN, USA) according to genes from the roots of soybean and corn with or without the manufacturer’s instructions. The purified products were LNA oligonucleotides were shown in Fig. 1. Mitochondria serially diluted and used for the 2nd run of amplification and plastid genes were predominantly and bacterial genes using primers 341F and 907R, which contained the A and were faintly observed in the PCR products amplified without B sequencing adapters (454 life sciences) (Appendix A). LNA oligonucleotides. In contrast, the bacterial genes were PCR reactions were performed in a 25-μL mixture that con- predominantly observed in the PCR products amplified with tained 12.5 μL premix hot start version (TaKaRa, Otsu, Ja- LNA oligonucleotides. This finding indicated that SSU rRNA pan), 1.0 μL A-341F and B-907R (20 pmol μL–1), and 1.0 μL genes of both soybean and corn organelles were prevented DNA template (20–50 ng of the template DNA). Sterilized from amplifying using LNA oligonucleotides. ultrapure water was added to a total volume of 25 μL. The following thermal program was used for amplification: 94°C 3.2. Root-associated bacterial diversity indices for 3 min (initial denaturation), followed by 30 cycles of 94°C for 1 min, 54°C for 1 min, and 72°C for 2 min, and finally, an 454 pyrosequencing technique was used to sequence extension at 72°C for 10 min. Each sample was amplified nested PCR products amplified with LNA and without LNA in triplicate, and the PCR products were pooled and purified oligonucleotides. After filtering poor-quality sequences, using the Agarose Gel DNA Purification Kit (TaKaRa, Dalian, 35 527 quality sequences with lengths of >400 bp from four China). An equal amount of the PCR product from each samples were obtained, and 5 317–13 109 sequences were sample was combined in a single tube and run on a Roche obtained per sample (mean=8 881), with an average of read FLX 454 pyrosequencing machine at Majorbio Bio-Pharm length of 518 bp. OTUs determination at 97% similarity 1886 YU Zhen-hua et al. Journal of Integrative Agriculture 2016, 15(8): 1883–1891

Negative LNA (–) LNA (+) M control Soybean Corn Soybean Corn Mitochondria

bp 1 500

1 000 Plastid Bacteria

Fig. 1 Agarose gel electrophoresis of the PCR products amplified from the roots of soybean and corn with or without LNA oligonucleotides. Lane M shows the 100-bp ladder. LNA (+), with locked nucleic acid (LNA) oligonucleotides; LNA (–), without LNA oligonucleotides. level were used to generate the rarefaction curve (Fig. 2) 800 Soybean LNA (+) and to calculate diversity indices of ACE, Chao1, Shannon, Soybean LNA (–) Corn LNA (+) Simpson and coverage (Table 1). Samples amplified with 600 Corn LNA (–) LNA oligonucleotides showed a higher number of OTUs, ACE, Chao1, Shannon indices, as well as lower Simpson 400 index than that of the corresponding samples without LNA oligonucleotides. For the soybean samples, no sequences No. of OTUs 200 were affiliated to plastids or mitochondria sequences when using LNA (+), whereas in using LNA (–), the proportion 0 of sequences derived from plastids and mitochondria was 66.43 and 1.33%, respectively. For the corn samples, 0 2 000 4 000 6 000 8 000 10 000 12 000 14 000 compared to LNA (–), the sequences belonging to plastids No. of reads sampled and mitochondria in using LNA (+) drastically decreased from 93.15 to 0.13% and from 4.23 to 0.08%, respectively. Fig. 2 Rarefaction analysis for the soybean and corn root samples. Curves were generated for 97% levels of operational 3.3. Taxonomic diversity of root-associated bacteria taxonomic unit (OTU).

To compare the taxonomic diversity among the four samples, dix B). Among them, Proteobacteria, Actinobacteria, and we randomly selected an equivalent number of sequences were the most abundant three phyla, which based on the minimum sequences (5 317) obtained from a corn LNA (+) sample. For bacterial taxonomic analysis, accounted for 47, 20, and 18% of the total sequences, re- the sequences of plastids and mitochondria were excluded. spectively. However, the high abundant soil bacterial phylum 129, 68, 159, and 45 genera affiliated to 14 phyla were ob- Acidobacteria was detected only 1%. The distribution of served in the samples of soybean LNA (+), soybean LNA root-associated bacteria in soybean LNA (+) and corn LNA (–), corn LNA (+), and corn LNA (–), respectively (Appen- (+) at phylum level was shown in Fig. 3.

Table 1 Richness and diversity indices of root-associated bacterial community in soybean and corn1) Number of Coverage Chloroplast Mitochondria Sample2) OTUs ACE Chao1 Shannon Simpson sequences (%) sequences (%) sequences (%) Soybean LNA (+) 13 109 465 824 722 3.57 0.09 97 0.00 0.00 Soybean LNA (–) 7 888 212 264 292 2.66 0.29 99 66.43 1.33 Corn LNA (+) 5 317 766 2 151 1 592 4.78 0.03 92 0.13 0.08 Corn LNA (–) 9 213 176 530 357 0.80 0.75 98 93.15 4.23 1) OTU, operational taxonomic unit, the number of phylotypes was calculated by sequences at the 97% similarity level. ACE, Chao1, Shannon, and Simpson, these indexes were calculated using MOTHUR program (http://www.mothur.org). 2) LNA (+), with locked nucleic acid (LNA) oligonucleotides; LNA (–), without LNA oligonucleotides. YU Zhen-hua et al. Journal of Integrative Agriculture 2016, 15(8): 1883–1891 1887

For the soybean samples, genera of Arthrobacter, the LNA (+) sample were Streptomyces (24%) and Niastella Bradyrhizobium, Erwinia, Flavobacterium, Methylotenera, (19%), which belonged to phyla Actinobacteria and Bacte- Pantoea, Polaromonas, Pseudomonas, and Variovorax roidetes, respectively. The percentage of other genus in the were only observed in the LNA (+) sample but not in the LNA LNA (+) sample was the range of 1–3%. Asteroleplasma, (–) sample. The genus Pseudomonas (31%), Bradyrhizobi- Bacillus, Chitinophaga, Flexibacter, Mucilaginibacter, Nias- um (21%) and Flavobacterium (14%) were the three most tella, Rhizobacter, and Rhodanobacter were only observed abundant genera, which belonged to phyla Proteobacteria in the corn LNA (+) sample, but not in LNA (–) sample. and Bacteroidetes. Although some genera, e.g., Burkholde- The root-associated bacterial composition between soy- ria, Massilia and Novosphingobium were only detected in bean and corn was compared using the LNA (+) samples. the LNA (–) sample and not in the LNA (+) sample, their The presence and absence of the major genera showed abundance were relatively low, which might have resulted that community composition between soybean and corn from stochastic PCR amplification or PCR biases. was distinctly different, and unique genera belonging to For the corn samples, the top two predominant genera in each crop were also identified (Fig. 4).

Acidobacteria (2.73%) Actinobacteria Others (1.03%) Firmicutes Others (1.73%) (5.60%) Bacteroidetes 8.37%

16.06% Proteobacteria 23.87% Actinobacteria 36.79% Proteobacteria Bacteroidetes 74.53% 29.28%

Soybean LNA (+) Corn LNA (+)

Fig. 3 Distribution of root-associated bacteria in soybean LNA (+) and corn LNA (+) at phylum level.

Soybean LNA (+) Corn LNA (+) Rhodanobacter Rhizobacter Niastella Mucilaginibacter Massilia Flexibacter Unique to corn Chitinophaga Burkholderia Bacillus Asteroleplasma Stenotrophomonas Polaromonas Rhizobium Pantoea Variovorax Methylotenera Unique to soybean Erwinia Flavobacterium Bradyrhizobium Pseudomonas Streptomyces Arthrobacter Shared

0102030 10 20 30

Fig. 4 Shared and unique genera between soybean LNA (+) and corn LNA (+). 1888 YU Zhen-hua et al. Journal of Integrative Agriculture 2016, 15(8): 1883–1891

A total of 12 OTUs from uncultured root-associated rarefaction curves generated from using LNA oligonucle- bacteria with >10 sequences were selected and subjected otides indicated higher levels of diversity of root-associated to BLAST analysis of NCBI (Table 2). All OTUs showed a bacterial community compared to earlier methodologies. low level of similarity to cultured bacteria and a high level The number of OTUs in the corn LNA (+) sample was higher of similarity to uncultured bacteria, which suggested that than that observed in the soybean LNA (+) sample, which certain uncharacterized bacteria inhabited the roots of may reflect that the corn sample supported a more diverse soybean and corn. root-associated bacterial community. However, additional studies are necessary to generate more evidence to support 4. Discussion the findings of the present study. In the soybean LNA (+) sample, Pseudomonas, Bradyrhi- The LNA oligonucleotide-PCR clamping combined with 454 zobium and Flavobacterium were the three most abundant pyrosequencing methods used in the present study provide genera. Pseudomonas was one of the most commonly a sufficient number of sequences of adequate length to isolated endophytic bacteria from a variety of plants such as estimate bacterial diversity and elucidate the compositions grapevine (West et al. 2010), roots of citrus trees (Gardner of root-associated bacterial communities. This method al- et al. 1982), roots of wheat (Germida and Siciliano 2001), po- lowed us to look in depth at microbial diversity with higher tato (Krechel et al. 2002) and corn (Montañez 2012). Many precision than those described previously (Chelius and of Pseudomonas spp. have showed anitimicrobial acitivity Triplett 2001; Hung et al. 2007; Ikenaga et al. 2015). The to plant pathogens (Dubeikovsky et al. 1993). The genus percentage of plastids and mitochondria in both soybean Bradyrhizobium can induce nitrogen-fixing nodules on the and corn LNA (+) samples was markedly reduced compared soybean roots. In addition, some members of Bradyrhizobi- to the corresponding LNA (–) samples, which confirmed that um can be candidates for plant growth-promoting rhizobac- the combined use of LNA-oligonucleotides with universal teria (Boiero et al. 2007; Masciarelli et al. 2014). Members of bacterial primers can prevent or inhibit PCR amplification Flavobacterium had been isolated from a number of diverse of plant organelle sequences. The diversity indices and habitats such as artificial lake (Kang et al. 2013), soil of a

Table 2 Closest relatives of uncultured root-associated bacteria derived from soybean and corn OTU No. of Seq Closest relative Similarity Source Alignment ID sequences (bp) Microorganisms1) Accession no. (%) 315 13 Corn 530 Uncultured Acidobacteria bacterium EF074678c 99 524/530 Anaeromyxobactersp. K NR_074969 83 445/533 318 20 Corn 543 Proteobacterium Ellin181 AJ292676 100 543/543 Steroidobacter denitrificans NR_044309 89 483/545 378 12 Corn 529 Uncultured Acidobacteria bacterium JQ861367 99 520/526 Candidatus Koribacterversatilis Ellin345 NR_074350 95 501/527 867 23 Corn/Soybean 525 Uncultured Gemmatimonadetes bacterium EU202844 100 525/525 Gemmatimonas aurantiacaT-27 NR_074708 86 456/529 1426 13 Corn 551 Uncultured bacterium HM125314 99 544/551 Gaiella occulta NR_118138 94 520/551 1429 99 Corn 527 Uncultured bacterium JQ357369 100 527/527 Leptothrix mobilis NR_026333 97 511/528 1761 117 Corn 500 Uncultured Flavobacteriaceae bacterium EF665357 99 499/500 Terrimonas lutea NR_041250 94 470/500 423 18 Corn 511 Uncultured bacterium EF072360 99 504/511 Niastella populi NR_116486 94 480/512 2294 21 Corn 517 Uncultured Sorangiineae bacterium FJ889275 99 515/517 Chondromyces lanuginosus NR_025345 93 482/517 946 19 Corn 509 Uncultured Methylococcaceae bacterium KC989528 95 484/509 Ectothiorhodospira variabilis NR_042700 88 442/500 1633 10 Corn 513 Uncultured bacterium JQ371697 97 454/467 Ectothiorhodospira magna NR_108987 89 446/503 2079 26 Corn 514 Uncultured Methylococcaceae bacterium EF019533 95 490/515 Ectothiorhodosinus mongolicus NR_042831 89 445/499 1) The first line shows the closest relative of bacteria from uncultured database and the 2nd line refers to the closest relative of bacteria from cultured database. YU Zhen-hua et al. Journal of Integrative Agriculture 2016, 15(8): 1883–1891 1889

ginseng field (Son et al. 2013), activated sludge (Park et al. monas and Rhizobium, which were detected in the present 2007), greenhouse soils (Kim et al. 2006) and terrestrial study, had also been reported to enhance the plant growth sample from the Antarctic (Yi et al. 2005). Some species (Gardner et al. 1982). These findings suggested that most of Flavobacterium can effectively protect plants against of the root-associated bacteria detected in the present study microbial infections by colonizing the roots (Sang and Kim could be potentially used in agricultural practices. 2012) or by bioremediation (Kang et al. 2013), whereas In the present study, some rare phylotypes were also de- some species were identified as plant-associated bacteria tected, although their abundance was negligible compared with potential beneficial effects for plant growth and health to the major bacterial sequences, some members were (Sessitsch et al. 2004). suspected to play specific roles in plant growth. For exam- For the corn LNA (+) sample, Streptomyces and Niastella ple, Enterobacter accounted for 0.49% of the total OTUs were the top two predominant genera. Streptomyces is in the soybean sample, which has been reported to have a biologically active plant-associated bacteria, has been nitrogen-fixing or antagonistic activity against phytopatho- isolated from a variety of plant tissues (Krechel et al. 2002; gens (Elbeltagy et al. 2001). Paenibacillus, a spore-forming Fialho de Oliveira et al. 2010). In addition, Streptomyces gram-positive bacteria, has been frequently isolated from induces a wide range of beneficial effects on the host rhizosphere soils and plant roots (Mavingui et al. 1992) and plant, which include antimicrobial activity, siderophore shown to play a variety of biological functions, including and indoleacetic acid production, and phosphate solubility biocontrol of plant diseases, nitrogen fixation and production (Fialho de Oliveira et al. 2010). Niastella is rarely reported of a wide variety of secondary metabolites, as well as plant as plant-associated bacteria or endophytic bacteria and cellwall-degrading enzymes (Gosalbes et al. 1991; Dijkster- a few species have been isolated to date. For example, huis et al. 1999; Chen et al. 2010). Several OTUs belonged was isolated from soil cultivated with to the group of uncultured bacteria, which indicated that the Korean ginseng (Weon et al. 2006), and Niastell apopuli soybean and corn root-associated bacterial communities was isolated from the soil of a Euphrates poplar (Populus are highly complex and their potential taxonomic affiliate euphratica) forest in Xinjiang, China (Zhang et al. 2010). information could be utilized in the development of isolation To our knowledge, information on the biological functions procedures for novel bacteria in further functional analysis. of Niastella is currently limited. In this study, we found the relative abundance of the 5. Conclusion major genera varied between soybean and corn, and unique genera belonging to each crop were also identified (Fig. 3), In summary, the present study is a pilot research that uti- which suggested that the root-associated bacterial compo- lized LNA-PCR clamping and pyrosequencing methods to sition of two crops in Mollisols was distinct. Germida and characterize root-associated bacterial communities in soy- Siciliano (2001) found that even in the same plant species bean and corn. Higher bacterial diversity was observed in (wheat), the bacterial endophytic community of the modern samples using LNA oligonucleotides than no using, which wheat cultivars was more diverse than that of the ancient further confirmed the advantage of using the LNA technique ones. Rijavec et al. (2007) isolated the bacterial endophytes in investigating plant-associated bacteria. The majority of from germinated maize kernels and also found genus the genera were distinctly different between soybean LNA Pantoea was associated with a specific maize cultivar. As (+) and corn LNA (+) root samples, which suggested differ- plants have a determinant role in controlling endophytic col- ent root-associated bacterial communities were selected onization (Chen et al. 2010), thus it is not surprising that the by two crops. Several uncultured root-associated bacteria dominant or unique groups of root-associated bacteria were detected in this study gives the clues for selecting the optimal different between soybean and corn. Since we prepared root medium and method to isolate the novel bacteria for further samples only once from the soybean-corn rotation system, functional analysis. further investigation of the temporal or seasonal fluctuations of root-associated bacterial community will provide more Acknowledgements valuable information on the ecological dynamics of the crop rotation system. This work was supported in part by grants from the Strategic Arthrobacter was detected in both soybean LNA (+) Priority Research Program of Chinese Academy of Sciences and corn LNA (+) root samples, but their proportion varied (XDB15010103) and the National Natural Science Founda- (Fig. 3). Arthrobacter, which is an endophytic bacteria tion of China (41201247). commonly isolated from corn, potato and other plants root samples, plays an important role in plant growth promotion Appendix associated with this paper can be available on (Krechel et al. 2002). Some species of Bacillus, Pseudo- http://www.ChinaAgriSci.com/V2/En/appendix.htm 1890 YU Zhen-hua et al. Journal of Integrative Agriculture 2016, 15(8): 1883–1891

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