5 research outputs found

    Identification of Nitrogen-Fixing Bradyrhizobium Associated With Roots of Field-Grown Sorghum by Metagenome and Proteome Analyses

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    Sorghum (Sorghum bicolor) is cultivated worldwide for food, bioethanol, and fodder production. Although nitrogen fixation in sorghum has been studied since the 1970s, N2-fixing bacteria have not been widely examined in field-grown sorghum plants because the identification of functional diazotrophs depends on the culture method used. The aim of this study was to identify functional N2-fixing bacteria associated with field-grown sorghum by using “omics” approaches. Four lines of sorghum (KM1, KM2, KM4, and KM5) were grown in a field in Fukushima, Japan. The nitrogen-fixing activities of the roots, leaves, and stems were evaluated by acetylene reduction and 15N2-feeding assays. The highest nitrogen-fixing activities were detected in the roots of lines KM1 and KM2 at the late growth stage. Bacterial cells extracted from KM1 and KM2 roots were analyzed by metagenome, proteome, and isolation approaches and their DNA was isolated and sequenced. Nitrogenase structural gene sequences in the metagenome sequences were retrieved using two nitrogenase databases. Most sequences were assigned to nifHDK of Bradyrhizobium species, including non-nodulating Bradyrhizobium sp. S23321 and photosynthetic B. oligotrophicum S58T. Amplicon sequence and metagenome analysis revealed a relatively higher abundance (2.9–3.6%) of Bradyrhizobium in the roots. Proteome analysis indicated that three NifHDK proteins of Bradyrhizobium species were consistently detected across sample replicates. By using oligotrophic media, we purified eight bradyrhizobial isolates. Among them, two bradyrhizobial isolates possessed 16S rRNA and nif genes similar to those in S23321 and S58T which were predicted as functional diazotrophs by omics approaches. Both free-living cells of the isolates expressed N2-fixing activity in a semi-solid medium according to an acetylene reduction assay. These results suggest that major functional N2-fixing bacteria in sorghum roots are unique bradyrhizobia that resemble photosynthetic B. oligotrophicum S58T and non-nodulating Bradyrhizobium sp. S23321. Based on our findings, we discuss the N2-fixing activity level of sorghum plants, phylogenetic and genomic comparison with diazotrophic bacteria in other crops, and Bradyrhizobium diversity in N2 fixation and nodulation

    Synergistic N2-fixation and salt stress mitigation in soybean through dual inoculation of ACC deaminase-producing Pseudomonas and Bradyrhizobium

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    Abstract We investigated the potential dual application of two Bradyrhizobium strains (B. diazoefficiens USDA110 and B. ottawaense SG09) and plant growth-promoting bacteria, PGPB (Pseudomonas spp.: OFT2 and OFT5), to improve nodulation and N2-fixation in soybean plants. The growth-promoting effects of dual inoculation were observed on plant growth, physiology, and nodulation of soybean under normal conditions compared with plants individually inoculated with either USDA110 or SG09. Both OFT2 and OFT5 promoted N2-fixation by 11% and 56%, respectively, when dual inoculation with USDA110 and by 76% and 81%, respectively, when dual inoculation with SG09. Salinity stress significantly reduces soybean growth, physiology, nutrient uptake, nodulation, and N2-fixation. However, these adverse effects were attenuated by the dual inoculation of PGPB and rhizobia depending on the combination of inoculants. In particular, dual inoculation of PGPB with SG09 was more effective in enhancing the salt tolerance of soybean by reducing salt-induced ethylene production and improving nutrient uptake. However, no such effect was observed with the combined inoculation of USDA110 and OFT5. An effective symbiotic association between SG09 and two Pseudomonas bacteria can be considered a beneficial approach to improving the symbiotic efficiency of nodulation and mitigating salinity stress in soybeans

    Bradyrhizobium ottawaense efficiently reduces nitrous oxide through high nosZ gene expression

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    Abstract N2O is an important greenhouse gas influencing global warming, and agricultural land is the predominant (anthropogenic) source of N2O emissions. Here, we report the high N2O-reducing activity of Bradyrhizobium ottawaense, suggesting the potential for efficiently mitigating N2O emission from agricultural lands. Among the 15 B. ottawaense isolates examined, the N2O-reducing activities of most (13) strains were approximately five-fold higher than that of Bradyrhizobium diazoefficiens USDA110T under anaerobic conditions. This robust N2O-reducing activity of B. ottawaense was confirmed by N2O reductase (NosZ) protein levels and by mitigation of N2O emitted by nodule decomposition in laboratory system. While the NosZ of B. ottawaense and B. diazoefficiens showed high homology, nosZ gene expression in B. ottawaense was over 150-fold higher than that in B. diazoefficiens USDA110T, suggesting the high N2O-reducing activity of B. ottawaense is achieved by high nos expression. Furthermore, we examined the nos operon transcription start sites and found that, unlike B. diazoefficiens, B. ottawaense has two transcription start sites under N2O-respiring conditions, which may contribute to the high nosZ expression. Our study indicates the potential of B. ottawaense for effective N2O reduction and unique regulation of nos gene expression towards the high performance of N2O mitigation in the soil
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