title: Endophytic Bradyrhizobium spp. isolates from sugarcane obtained through different culture strategies.
abstract: 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.
authors.affiliation: Embrapa Agrobiologia, BR 465 km 7, 23891-460, Seropédica, Rio de Janeiro, Brazil.
authors.forename: Luc Felicianus Marie
authors.initials: LF
authors.lastname: Rouws
authors.forename: Jakson
authors.initials: J
authors.lastname: Leite
authors.forename: Gustavo Feitosa
authors.initials: GF
authors.lastname: de Matos
authors.forename: Jerri Edson
authors.initials: JE
authors.lastname: Zilli
authors.forename: Marcia Reed Rodrigues
authors.initials: MR
authors.lastname: Coelho
authors.forename: Gustavo Ribeiro
authors.initials: GR
authors.lastname: Xavier
authors.forename: Doreen
authors.initials: D
authors.lastname: Fischer
authors.forename: Anton
authors.initials: A
authors.lastname: Hartmann
authors.forename: Verônica Massena
authors.initials: VM
authors.lastname: Reis
authors.forename: José Ivo
authors.initials: JI
authors.lastname: Baldani
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D020369:Bradyrhizobium / Q000145:classification* / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D001938:Brazil; D016000:Cluster Analysis; D003470:Culture Media / Q000737:chemistry; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D060026:Endophytes / Q000145:classification* / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D008828:Microbiological Techniques; D008969:Molecular Sequence Data; D058889:Molecular Typing; D009591:Nitrogenase / Q000032:analysis; D010802:Phylogeny; D055170:Plant Root Nodulation; D018517:Plant Roots / Q000382:microbiology; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D031786:Saccharum / Q000382:microbiology*; D017422:Sequence Analysis, DNA
chemical_list: D001426:Bacterial Proteins; D003470:Culture Media; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S; D009591:Nitrogenase