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

Abstract

Aims Previous studieshaveshownthatelephantgrassis 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 RNAwere 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 RNAderivedDGGE-profilesdiffered,especiallywithintissue 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 RNAderived 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