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

• Published in: Plant and soil 2013-12, Vol.373 (1-2), p.737-754
• Main Authors: Videira, Sandy Sampaio, Pereira e Silva, Michele de Cássia, de Souza Galisa, Péricles, Dias, Armando Cavalcante Franco, Nissinen, Riitta, Divan, Vera Lúcia Baldani, van Elsas, Jan Dirk, Baldani, José Ivo, Salles, Joana Falcão
• Format: Article
• Language: English
• Subjects: Agricultural research; Agricultural soils; Agronomy. Soil science and plant productions; Animal, plant and microbial ecology; Bacteria; bacterial communities; Biological and medical sciences; Biological diversity; Biomedical and Life Sciences; Bradyrhizobium; Burkholderia; clones; complementary DNA; denaturing gradient gel electrophoresis; Deoxyribonucleic acid; DNA; Ecology; Elephants; Endophytes; Energy crops; Environmental aspects; Fundamental and applied biological sciences. Psychology; General agronomy. Plant production; genes; genotype; Genotype & phenotype; Genotypes; Grasses; in vitro culture; Leptothrix (bacteria); Libraries; Life Sciences; Nitrogen fixation; nitrogen-fixing bacteria; Nitrogen-fixing microorganisms; nucleotide sequences; Pennisetum purpureum; phylogeny; Plant extracts; Plant Physiology; Plant roots; Plant Sciences; Plant tissues; Plants; Polymerase chain reaction; Regular Article; rhizoplane; ribosomal RNA; Roots; Soil microorganisms; Soil Science & Conservation; Soil-plant relationships. Soil fertility; Soil-plant relationships. Soil fertility. Fertilization. Amendments; species diversity; Stems; Sustainable production
• ISSN: 0032-079X; 1573-5036
• DOI: 10.1007/s11104-013-1828-4

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.