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A Culture-Independent Approach to Enrich Endophytic Bacterial Cells from Sugarcane Stems for Community Characterization

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Abstract

Bacterial endophytes constitute a very diverse community and they confer important benefits which help to improve agricultural yield. Some of these benefits remain underexplored or little understood, mainly due to the bottlenecks associated with the plant feature, a low number of endophytic bacterial cells in relation to the plant, and difficulties in accessing these bacteria using cultivation-independent methods. Enriching endophytic bacterial cells from plant tissues, based on a non-biased, cultivation-independent physical enrichment method, may help to circumvent those problems, especially in the case of sugarcane stems, which have a high degree of interfering factors, such as polysaccharides, phenolic compounds, nucleases, and fibers. In the present study, an enrichment approach for endophytic bacterial cells from sugarcane lower stems is described. The results demonstrate that the enriched bacterial cells are suitable for endophytic community characterization. A community analysis revealed the presence of previously well-described but also novel endophytic bacteria in sugarcane tissues which may exert functions such as plant growth promotion and biological control, with a predominance of the Proteobacterial phylum, but also Actinobacteria, Bacteroidetes, and Firmicutes, among others. In addition, by comparing the present and literature data, it was possible to list the most frequently detected bacterial endophyte genera in sugarcane tissues. The presented enrichment approach paves the way for improved future research toward the assessment of endophytic bacterial community in sugarcane and other biofuel crops.

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Acknowledgements

This work has been funded by The National Council for Scientific and Technological Development (CNPq—“National Institute of Science and Technology of Biological Nitrogen Fixation”, grant no. 573.828/2008-3), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (Faperj—C.M.S. “Iniciação Científica” fellowship, no. E-26/102.098/2012, and grants “Desenvolvimento Científico e Tecnológico Regional”, no. E-26/110.235/2011, and “Auxílio à Pesquisa APQ1”, no. E-26/111.574/2013), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES—C.M.S. doctorate fellowship, call CAPES/Embrapa no. 015/2014), and Brazilian Agricultural Research Corporation (Embrapa). We thank the supports of Daniella Pessoa for reviewing the English writing of this manuscript and Michelle Z. T. Sfeir and Valter A. de Baura (Universidade Federal do Paraná) for helping on Illumina sequencing of the samples.

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Correspondence to Stefan Schwab.

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Online Resource 1

Micrograph of the reporter bacterium in the Neubauer chamber used to count cells. Scale bars: 10 μm (GIF 116 kb)

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Optimized classification of OTUs into species, their plant growth-promotion functions according to literature data, and from which tissues they were previously detected in, or isolated from, the endosphere of sugarcane. All data are normalized (XLSX 33 kb)

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Data compilation to define the most quoted endophytic bacterial genera group in sugarcane tissues. All previously published data used have been obtained through 16S rRNA gene sequencing analysis of surface-disinfected sugarcane tissues (leaves, roots, and/or stems) (XLSX 26.4 kb)

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Dos-Santos, C.M., de Souza, D.G., Balsanelli, E. et al. A Culture-Independent Approach to Enrich Endophytic Bacterial Cells from Sugarcane Stems for Community Characterization. Microb Ecol 74, 453–465 (2017). https://doi.org/10.1007/s00248-017-0941-y

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  • DOI: https://doi.org/10.1007/s00248-017-0941-y

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