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Journal of Integrative Agriculture  2016, Vol. 15 Issue (8): 1883-1891    DOI: 10.1016/S2095-3119(15)61195-9
Soil & Fertilization﹒Irrigation﹒Plant Nutrition﹒ Agro-Ecology & Environment Advanced Online Publication | Current Issue | Archive | Adv Search |
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
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Abstract      The community structure and diversity of root-associated bacteria 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 without 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 Niastella. 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:

This work was supported in part by grants from the Strategic Priority Research Program of Chinese Academy of Sciences (XDB15010103) and the National Natural Science Foundation of China (41201247).

Corresponding Authors:  WANG Guang-hua, Tel: +86-451-86602745, Fax: +86-451-86603736, E-mail:; Makoto Ikenaga, Tel: +81-99-2858660, E-mail:   

Cite this article: 

YU Zhen-hua, YU Jiang, Makoto Ikenaga, Masao Sakai, LIU Xiao-bing, WANG Guang-hua. 2016. Characterization of root-associated bacterial community structures in soybean and corn using locked nucleic acid (LNA) oligonucleotide- PCR clamping and 454 pyrosequencing. Journal of Integrative Agriculture, 15(8): 1883-1891.

Acosta-Martinez V, Dowd S, Sun Y, Allen V. 2008. Tag-encoded pyrosequencing analysis of bacterial diversity in a single soil type as affected by management and land use. Soil Biology and Biochemistry, 40, 2762–2770.

Bashan Y, Holguin G, de-Bashan L E. 2004. Azospirillum-plant relationships: Physiological, molecular, agricultural, and environmental advances (1997–2003). Canadian Journal of Microbiology, 50, 521–577. 

Boiero L, Perrig D, Masciarelli O, Penna C, Cassán F, Luna V. 2007. Phytohormone production by three strains of Bradyrhizobium japonicum and possible physiological and technological implications. Applied Microbiology and Biotechnology, 74, 874–880.

Chelius M, Triplett E. 2001. The diversity of archaea and bacteria in association with the roots of Zea mays L. Microbial Ecology, 41, 252–263.

Chen X L, Wang G H, Xu M N, Jin J, Liu X B. 2010. Antifungal peptide produced by Paenibacilluspolymyxa BRF-1 isolated from soybean rhizosphere. African Journal of Microbiology Research, 4, 2692–2698.

Dijksterhuis J, Sanders M, Gorris L, Smid E. 1999. Antibiosis plays a role in the context of direct interaction during antagonism of Paenibacillus polymyxa towards Fusarium oxysporum. Journal of Applied Microbiology, 86, 13–21.

Dubeikovsky A N, Mordukhova E A, Kochetkov V V, Polikarpova F Y, Boronin A M. 1993. Growth promotion of blackcurrant softwood cuttings by recombinant strain Pseudomonas fluorescens BSP53a synthesizing an increased amount of indole-3-acetic acid. Soil Biology and Biochemistry, 25, 1277–1281.

Elbeltagy A, Nishioka K, Sato T, Suzuki H, Ye B, Hamada T, Isawa T, Mitsui H, Minamisawa K. 2001. Endophytic colonization and in planta nitrogen fixation by a Herbaspirillum sp. isolated from wild rice species. Applied and Environmental Microbiology, 67, 5285–5293. 

Ferrando L, Mañay J F, Scavino A F. 2012. Molecular and culture-dependent analyses revealed similarities in the endophytic bacterial community composition of leaves from three rice (Oryza sativa) varieties. FEMS Microbiology Ecology, 80, 696–708.

Fialho de Oliveira M, Germano da Silva M, Van Der Sand S T. 2010. Anti-phytopathogen potential of endophytic actinobacteria isolated from tomato plants (Lycopersicon esculentum) in southern Brazil, and characterization of Streptomyces sp. R18 (6), a potential biocontrol agent. Research in Microbiology, 161, 565–572. 

Gardner J M, Feldman A W, Zablotowicz R M. 1982. Identity and behavior of xylem-residing bacteria in rough lemon roots of florida citrus trees. Applied and Environmental Microbiology, 43, 1335–1342.

Germaine K J, Liu X, Cabellos G G, Hogan J P, Ryan D, Dowling D N. 2006. Bacterial endophyte-enhanced phytoremediation of the organochlorine herbicide 2,4-dichlorophenoxyacetic acid. FEMS Microbiology Ecology, 57, 302–310. 

Germida J, Siciliano S. 2001. Taxonomic diversity of bacteria associated with the roots of modern, recent and ancient wheat cultivars. Biology and Fertility of Soils, 33, 410–415. 

Gosalbes M J, Pérez-González J A, Gonzalez R, Navarro A. 1991. Two beta-glycanase genes are clustered in Bacillus polymyxa: molecular cloning, expression, and sequence analysis of genes encoding a xylanase and an endo-beta-(1,3)-(1,4)-glucanase. Journal of Bacteriology, 173, 7705–7710. 

Hallmann J, Quadt-Hallmann A, Mahaffee W F, Kloepper J W. 1997. Bacterial endophytes in agricultural crops. Canadian Journal of Microbiology, 43, 895–914.

Hung P, Kumar S, Govindsamy V, Annapurna K. 2007. Isolation and characterization of endophytic bacteria from wild and cultivated soybean varieties. Biology and Fertility of Soils, 44, 155–162.

Ikenaga M, Sakai M. 2014. Application of locked nucleic acid (LNA) oligonucleotide-PCR clamping technique to selectively PCR amplify the SSU rRNA genes of bacteria in investigating the plant-associated community structures. Microbes and Environments, 29, 286–295.

Ikenaga M, Tabuchi M, Oyama T, Akagi I, Sakai M. 2015. Development of LNA oligonucleotide-PCR clamping technique in investigating the community structures of plant-associated bacteria. Bioscience, Biotechnology, and Biochemistry, 79, 1556–1566.

Kang J Y, Chun J, Jahng K Y. 2013. Flavobacterium aciduliphilum sp. nov., isolated from freshwater, and emended description of the genus Flavobacterium. International Journal of Systematic and Evolutionary Microbiology, 63, 1633–1638.

Kim B Y, Weon H Y, Cousin S, Yoo S H, Kwon S W, Go S J, Stackebrandt E. 2006. Flavobacterium daejeonense sp. nov. and Flavobacterium suncheonense sp. nov., isolated from greenhouse soils in Korea. International Journal of Systematic and Evolutionary Microbiology, 56, 1645–1649.

Krechel A, Faupel A, Hallmann J, Ulrich A, Berg G. 2002. Potato-associated bacteria and their antagonistic potential towards plant-pathogenic fungi and the plant-parasitic nematode Meloidogyne incognita (Kofoid & White) Chitwood. Canadian Journal of Microbiology, 48, 772–786.

Lane D J. 1991. 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M, eds., Nucleic Acid Techniques in Bacterial Systematics. John Wiley & Sons, New York. pp. 115–175.

Li Y H, Liu Q F, Liu Y, Zhu J N, Zhang Q. 2011. Endophytic bacterial diversity in roots of Typha angustifolia L. in the constructed Beijing Cuihu Wetland (China). Research in Microbiology, 162, 124–131.

Mano H, Tanaka F, Watanabe A, Kaga H, Okunishi S, Morisaki H. 2006. Culturable surface and endophytic bacterial flora of the maturing seeds of rice plants (Oryza sativa) cultivated in a paddy field. Microbes and Environments, 21, 86–100.

Masciarelli O, Llanes A, Luna V. 2014. A new PGPR co-inoculated with Bradyrhizobium japonicum enhances soybean nodulation. Microbiological Research, 169, 609–615.

Mavingui P, Laguerre G, Berge O, Heulin T. 1992. Genetic and phenotypic diversity of Bacillus polymyxa in soil and in the wheat rhizosphere. Applied and Environmental Microbiology, 58, 1894–1903.

Mazzaglia A, Anselmi N, Gasbarri A, VanniniA. 2001. Development of a polymerase chain reaction (PCR) assay for the specific detection of Biscogniauxia mediterranea living as an endophyte in oak tissues. Mycological Research, 105, 952–956.

Montañez A, Blanco A R, Barlocco C, Beracochea M, Sicardi M. 2012. Characterization of cultivable putative endophytic plant growth promoting bacteria associated with maize cultivars (Zea mays L.) and their inoculation effects in vitro. Applied Soil Ecology, 58, 21–28.

Park M, Ryu S H, Vu T H T, Ro H S, Yun P Y, Jeon C O. 2007. Flavobacterium defluvii sp. nov., isolated from activated sludge. International Journal of Systematic and Evolutionary Microbiology, 57, 233–237.

Peay K G, Baraloto C, Fine P V. 2013. Strong coupling of plant and fungal community structure across western Amazonian rainforests. The ISME Journal, 7, 1852–1861.

Rastogi G, Tech J J, Coaker G L, Leveau J H J. 2010. A PCR-based toolbox for the culture-independent quantification of total bacterial abundances in plant environments. Journal of Microbiological Methods, 83, 127–132.

Rijavec T, Lapanje A, Dermastia M, Rupnik M. 2007. Isolation of bacterial endophytes from germinated maize kernels. Canadian Journal of Microbiology, 53, 802–808.

Ronaghi M. 2001. Pyrosequencing sheds light on DNA sequencing. Genome Research, 11, 3–11.

Sang M K, Kim K D. 2012. The volatile-producing Flavobacterium johnsoniae strain GSE09 shows biocontrol activity against Phytophthora capsici in pepper. Journal of Applied Microbiology, 113, 383–398.

Sessitsch A, Reiter B, Berg G. 2004. Endophytic bacterial communities of field-grown potato plants and their plant-growth-promoting and antagonistic abilities. Canadian Journal of Microbiology, 50, 239–249.

Son H M, Kook M, Park S Y, Mavlonov G T, Yi T H. 2013. Flavobacterium kyungheensis sp. nov., isolated from soil of a ginseng field. Antonie van Leeuwenhoek, 104, 1029–1037.

Sun L, Qiu F, Zhang X, Dai X, Dong X, Song W. 2008. Endophytic bacterial diversity in rice (Oryza sativa L.) roots estimated by 16S rDNA sequence analysis. Microbial Ecology, 55, 415–424.

Tholozan J L, Cappelier J M, Tissier J P, Delattre G, Federighi M. 1999. Physiological characterization of viable-but-nonculturable Campylobacter jejuni cells. Applied and Environmental Microbiology, 65, 1110–1116.

Weon H Y, Kim B Y, Yoo S H, Lee S Y, Kwon S W, Go S J, Stackebrandt E. 2006. Niastella koreensis gen. nov., sp. nov. and Niastella yeongjuensis sp. nov., novel members of the phylum Bacteroidetes, isolated from soil cultivated with Korean ginseng. International Journal of Systematic and Evolutionary Microbiology, 56, 1777–1782.

West E, Cother E, Steel C, Ash G. 2010. The characterization and diversity of bacterial endophytes of grapevine. Canadian Journal of Microbiology, 56, 209–216.

Yi H, Oh H M, Lee J H, Kim S J, Chun J. 2005. Flavobacterium antarcticum sp. nov., a novel psychrotolerant bacterium isolated from the Antarctic. International Journal of Systematic and Evolutionary Microbiology, 55, 637–641.

Zhang K, Wang Y, Tang Y, Dai J, Zhang L, An H, Luo G, Rahman E, Fang C. 2010. Niastella populi sp. nov., isolated from soil of Euphrates poplar (Populus euphratica) forest, and emended description of the genus Niastella. International Journal of Systematic and Evolutionary Microbiology, 60, 542–545.
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