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Endophytic Bradyrhizobium spp. isolates from sugarcane obtained through different culture strategies

Rouws, Luc Felicianus Marie, Leite, Jakson, Matos, Gustavo Feitosa, Zilli, Jerri Edson, Coelho, Marcia Reed Rodrigues, Xavier, Gustavo Ribeiro, Fischer, Doreen, Hartmann, Anton, Reis, Verônica Massena, Baldani, José Ivo
Environmental microbiology reports 2014 v.6 no.4 pp. 354-363
Bradyrhizobium, Vigna unguiculata, acetylene reduction, bacteria, complementary DNA, cultivars, culture media, endophytes, genes, legumes, nitrogen, nitrogen fixation, nitrogenase, polymerase chain reaction, repetitive sequences, ribosomal RNA, roots, sequence analysis, sugarcane
Brazilian sugarcane has been shown to obtain part of its nitrogen via biological nitrogen fixation (BNF). Recent reports, based on the culture independent sequencing of bacterial nifH complementary DNA (cDNA) from sugarcane tissues, have suggested that members of the Bradyrhizobium genus could play a role in sugarcane‐associated BNF. Here we report on the isolation of Bradyrhizobium spp. isolates and a few other species from roots of sugarcane cultivar RB867515 by two cultivation strategies: direct isolation on culture media and capture of Bradyrhizobium spp. using the promiscuous legume Vigna unguiculata as trap‐plant. Both strategies permitted the isolation of genetically diverse Bradyrhizobium spp. isolates, as concluded from enterobacterial repetitive intergenic consensus polymerase chain reaction (PCR) fingerprinting and 16S ribosomal RNA, nifH and nodC sequence analyses. Several isolates presented nifH phylotypes highly similar to nifH cDNA phylotypes detected in field‐grown sugarcane by a culture‐independent approach. Four isolates obtained by direct plate cultivation were unable to nodulate V. unguiculata and, based on PCR analysis, lacked a nodC gene homologue. Acetylene reduction assay showed in vitro nitrogenase activity for some Bradyrhizobium spp. isolates, suggesting that these bacteria do not require a nodule environment for BNF. Therefore, this study brings further evidence that Bradyrhizobium spp. may play a role in sugarcane‐associated BNF under field conditions.