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Culture-independent molecular approaches reveal a mostly unknown high diversity of active nitrogen-fixing bacteria associated with Pennisetum purpureum—a bioenergy crop

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Abstract

Aims

Previous studies have shown that elephant grass is colonized by nitrogen-fixing bacterial species; however, these results were based on culture-dependent methods, an approach that introduces bias due to an incomplete assessment of the microbial community. In this study, we used culture-independent methods to survey the diversity of endophytes and plant-associated bacterial communities in five elephant grass genotypes used in bioenergy production.

Methods

The plants of five genotypes of elephant grass were harvested from the experimental area of Embrapa Agrobiologia and divided into stem and root tissues. Total DNA and RNA were extracted from plant tissues and the bacterial communities were analyzed by DGGE and clone library of the 16S rRNA and nifH genes at both the cDNA and DNA levels.

Results

Overall, the patterns based on DNA- and RNA-derived DGGE-profiles differed, especially within tissue samples. DNA-based DGGE indicated that both total bacterial and diazotrophic communities associated with roots (rhizoplane + endophytes) differed clearly from those obtained from stems (endophytes). These results were confirmed by the phylogenetic analyses of RNA-derived sequences of 16S rRNA (total bacteria; 586 sequences), but not for nifH (186). In fact, rarefaction analyses showed a higher diversity of diazotrophic organisms associated with stems than roots. Based on 16S rRNA sequences, the clone libraries were dominated by sequences affiliated to members of Leptotrix (12.8 %) followed by Burkholderia (9 %) and Bradyrhizobium (6.5 %), while most of the nifH clones were closely related to the genus Bradyrhizobium (26 %).

Conclusions

Our results revealed an unexpectedly large diversity of metabolically active bacteria, providing new insights into the bacterial species predominantly found in association with elephant grass. Furthermore, these results can be very useful for the development of new strategies for selection of potential bacteria that effectively contribute to biological nitrogen fixation and enhance the sustainable production of elephant grass as bioenergy crop.

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Acknowledgments

This work was part of the activities carried out at Department of Microbial Ecology, Center for Ecological and Evolutionary Studies, University of Groningen. The authors thank the support in the form of fellowships to SSV by Foundation for Research Support in the State of Rio de Janeiro (FAPERJ) and thankfully acknowledged Embrapa and CNPq/INCT-FBN for partial financial support.

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Authors and Affiliations

  1. Universidade Federal Rural do Rio de Janeiro (UFRRJ), Rio de Janeiro, Brazil

    Sandy Sampaio Videira & Péricles de Souza Galisa

  2. Centro Universitário de Volta Redonda (UniFOA), Rio de Janeiro, Brazil

    Sandy Sampaio Videira

  3. Department of Microbial Ecology, Center for Ecological and Evolutionary Studies (CEES), University of Groningen (RUG), Nijenborgh 7, 9747AG, Groningen, The Netherlands

    Michele de Cássia Pereira e Silva, Riitta Nissinen, Jan Dirk van Elsas & Joana Falcão Salles

  4. Center for Nuclear Energy in Agriculture, Universidade de São Paulo, CENA/USP, Piracicaba, Brazil

    Armando Cavalcante Franco Dias

  5. Embrapa Agrobiologia (CNPAB), BR 465, km 07, CEP 23890-000, Seropédica, Rio de Janeiro, Brazil

    Sandy Sampaio Videira, Péricles de Souza Galisa, Vera Lúcia Baldani Divan & José Ivo Baldani

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  1. Sandy Sampaio Videira
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  2. Michele de Cássia Pereira e Silva
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  3. Péricles de Souza Galisa
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  5. Riitta Nissinen
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  6. Vera Lúcia Baldani Divan
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  7. Jan Dirk van Elsas
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  8. José Ivo Baldani
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  9. Joana Falcão Salles
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Correspondence to Joana Falcão Salles.

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Responsible Editor: Katharina Pawlowski.

In memoriam (Péricles de Souza Galisa)

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Figure S1

Denaturing gradient gel electrophoresis (DGGE) profile of 16S rDNA (A) and rRNA (B) from communities associated with root and stem tissues of 5 elephant grass genotypes. Three replicates were used per sample. The samples included in these figures are taken from root (R) and stem (S) of genotype Cameroon (G1), Gramafante (G2); BAG 02 (G3), Roxo (G4), CNPGL91F06-3 (G5) and M represent DGGE marker. (PPT 691 kb)

Figure S2

Denaturing gradient gel electrophoresis (DGGE) profile of nifH gene fragments from DNA (A) and RNA (B) pools obtained from root and stem tissues of 5 elephant grass genotypes. Three replicates were used per sample. The samples included in these figures are taken from root (R) and stem (S) of genotype Cameroon (G1), Gramafante (G2); BAG 02 (G3), Roxo (G4), CNPGL91F06-3 (G5) and M represent DGGE marker. (PPT 637 kb)

Figure S3

Venn diagrams based on the 16S rRNA gene libraries from root (A), stem (B) and all sequences together (C) showing the intersections and peculiarities among tree elephant grass genotypes including G1- Cameroon, G4- Roxo e G5- CNPGL91F06-3. (PPT 113 kb)

Table S1

Information for elephant grass genotypes (cultivars) used in the experiments. The genotypes are represented in the following format: Cameroon (G1), Gramafante (G2), BAG 02 (G3), Roxo (G4), and CNPGL91F06-3 (G5). (PPT 87 kb)

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Videira, S.S., Pereira e Silva, M.d.C., de Souza Galisa, P. et al. Culture-independent molecular approaches reveal a mostly unknown high diversity of active nitrogen-fixing bacteria associated with Pennisetum purpureum—a bioenergy crop. Plant Soil 373, 737–754 (2013). https://doi.org/10.1007/s11104-013-1828-4

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  • DOI: https://doi.org/10.1007/s11104-013-1828-4

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