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Abstract
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.
Highlights
Biological N2 fixation in non-leguminous plants is required to improve agricultural sustainability by decreasing the global use of synthetic nitrogen fertilizers (Steffen et al, 2015; Yoneyama et al, 2017; Rosenblueth et al, 2018)
We identified tissues showing significant N2-fixing activity by acetylenereducing activity (ARA) and 15N2 fixation, identified functional diazotrophs by proteome analysis of nitrogenase proteins based on metagenomic data, and isolated bacteria with nitrogenase proteins and phylogenetic markers predicted from the omics results (Figure 1)
Our results suggest that sorghum roots harbor functional N2-fixing bradyrhizobia that are similar to photosynthetic B. oligotrophicum S58T and Bradyrhizobium sp
Summary
Biological N2 fixation in non-leguminous plants is required to improve agricultural sustainability by decreasing the global use of synthetic nitrogen fertilizers (Steffen et al, 2015; Yoneyama et al, 2017; Rosenblueth et al, 2018). Diazotrophic endophytes can provide fixed nitrogen to non-leguminous crops (Boddey et al, 2001; Yoneyama et al, 2017). 15N-labeling significantly contributed to N2 fixation in sugarcane (Yoneyama et al, 1997; Boddey et al, 2001). Recent metatranscriptome analyses targeting nifH (encoding dinitrogenase reductase) suggested that Bradyrhizobium members play a role in N2 fixation in sugarcane (Thaweenut et al, 2011; Fischer et al, 2012; Rosenblueth et al, 2018). Sorghum root-associated microbiomes play an important role in determining plant fitness
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