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.

Segregation of nod‐containing and nod‐deficient bradyrhizobia as endosymbionts of Arachis hypogaea and as endophytes of Oryza sativa in intercropped fields of Bengal Basin, India

“…All WBOS isolates were clustered in BD clade along with several natural bradyrhizobial endophytes of sugarcane like AG48, AG41, P4‐7, P9‐20 and P4‐29 (Rouws et al ., ), as well as those of cultivated rice from Thailand like SUT‐PR48, SUT‐PR64, SUT‐PR9, SUT‐R55, SUT‐R74 (Piromyou et al ., ). WBOS7 was an exception which clustered in BJ with the WBAH strains.…”

“…The free living nitrogen‐fixing activity of the bacterial cultures was examined by Acetylene Reduction Assay as described by Rouws et al ., . The reaction was carried out in JMV medium (Reis et al ., ) supplemented with 0.4 g l −1 yeast extract and LG medium supplemented with arabinose and 0.001% yeast extract (Noisangiam et al ., ).…”

“…The free living nitrogen-fixing activity of the bacterial cultures was examined by Acetylene Reduction Assay as described by Rouws et al, 2014. The reaction was carried out in JMV medium (Reis et al, 2004) supplemented with 0.4 g l 21 yeast extract and LG medium supplemented with arabinose and 0.001% yeast extract (Noisangiam et al, 2012). Serum vials (10 ml) were filled with 5 ml semi-solid media, inoculated with 50 ml cell suspensions in sterile Milli-Q water, sealed and incubated at 288C for 7 days.…”

“…'Crack invasion' draws parallel with the mode of endophytic colonization in the non-legumes (Rouws et al, 2014). Intriguingly in several cases, Bradyrhizobium endosymbionts of Aeschynomene legumes have been found to colonise in different Oryza cultivars as endophytes by invading through epidermal cracks.…”

The Influence of Land Use Intensity on the Plant-Associated Microbiome of Dactylis glomerata L.

“…Numerous families within Rhizobiales , e.g., Rhizobiaceae , are known for their plant-growth promoting and nitrogen-fixing abilities in Fabaceae (Patriarca et al, 2002; Chi et al, 2005). However, members of Rhizobiales were also found to express nifH in roots of sugarcane, a gene which encodes enzymes involved in biological nitrogen fixation (Rouws et al, 2014), thus enhancing plant performance under limited nitrogen availability. These findings indicate that Rhizobiales also play an important role in perennial grasses, albeit without nodule formation.…”