Kiwamu Minamisawa

Diverse members of Bradyrhizobium diazoefficiens, B. japonicum, and B. ottawaense were isolated from the roots of field-grown sorghum plants in Fukushima, and classified into “Rhizobia” with nodulated soybeans, “Free-living diazotrophs”, and “Non-diazotrophs” by nitrogen fixation and nodulation assays. Genome analyses revealed that B. ottawaense members possessed genes for N2O reduction, but lacked those for the Type VI secretion system (T6SS). T6SS is a new bacterial weapon against microbial competitors. Since T6SS-possessing B. diazoefficiens and B. japonicum have mainly been isolated from soybean nodules in Japan, T6SS-lacking B. ottawaense members may be a cryptic lineage of soybean bradyrhizobia in Japan.

Biological nitrogen fixation (BNF) by plants and its bacterial associations represent an important natural system for capturing atmospheric dinitrogen (N₂) and processing it into a reactive form of nitrogen through enzymatic reduction. The study of BNF in non-leguminous plants has been difficult compared to nodule-localized BNF in leguminous plants because of the diverse sites of N₂ fixation in non-leguminous plants. Identification of the involved N₂-fixing bacteria has also been difficult because the major nitrogen fixers were often lost during isolation attempts. The past 20 years of molecular analyses has led to the identification of N₂ fixation sites and active nitrogen fixers in tissues and the rhizosphere of non-leguminous plants. Here, we examined BNF hotspots in six reported non-leguminous plants. Novel rhizobia and methanotrophs were found to be abundantly present in the free-living state at sites where carbon and energy sources were predominantly available. In the carbon-rich apoplasts of plant tissues, rhizobia such as Bradyrhizobium spp. microaerobically fix N₂. In paddy rice fields, methane molecules generated under anoxia are oxidized by xylem aerenchyma-transported oxygen with the simultaneous fixation of N₂ by methane-oxidizing methanotrophs. We discuss the effective functions of the rhizobia and methanotrophs in non-legumes for the acquisition of fixed nitrogen in addition to research perspectives.

Nitrous oxide (N2O) is the third most important greenhouse gas after carbon dioxide and methane, and a prominent ozone-depleting substance. Agricultural soils are the primary anthropogenic source of N2O because of the constant increase in the use of industrial nitrogen (N) fertilizers. The soybean crop is grown on 6% of the world’s arable land, and its production is expected to increase rapidly in the future. In this review, we summarize the current knowledge on N-cycle in the rhizosphere of soybean plants, particularly sources and sinks of N2O. Soybean root nodules are the host of dinitrogen (N2)-fixing bacteria from the genus Bradyrhizobium. Nodule decomposition is the main source of N2O in soybean fields, and both bradyrhizobia and other rhizospheric microorganisms are responsible for the nitrogen transformations that produce N2O. This N2O is either emitted into the atmosphere or further reduced to N2 by the bradyrhizobial N2O reductase (N2OR), encoded by the nos gene cluster. The dominance of nos− indigenous populations of soybean bradyrhizobia results in the emission of N2O into the atmosphere. Hence, inoculation with nos+ or nos++ (mutants with enhanced N2OR activity) bradyrhizobia has proved to be promising strategies to reduce N2O emission in the field. We discussed these strategies, the molecular mechanisms underlying them, and the future perspectives to develop better options for global mitigation of N2O emission from soils.

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 S58^T. 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 S58^T 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 S58^T 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.

We report the complete genome sequence of Bradyrhizobium diazoefficiens USDA 122, a nitrogen-fixing soybean symbiont. The genome consists of a 9.1 Mb circular chromosome, and 8,551 coding sequences (CDSs) were predicted on the genome. The sequence will provide insight into the evolution of rhizobial genome, and the symbiotic compatibility with host plants.

Soybean bradyrhizobia form root nodules on soybean plants and symbiotically fix N ₂ . Strain J5 is phylogenetically far from well-known representatives within the Bradyrhizobium japonicum linage. The complete genome showed the largest single chromosomal (10.1 Mb) and symbiosis island (998 kb) among complete genomes of soybean bradyrhizobia.

N ₂ -fixing methanotrophs play an important role in the methane-nitrogen cycle in rice paddies. We report here the draft genome sequence of Methylosinus sp. strain 3S-1 isolated from rice root in a paddy field without N fertilizer input.

Agricultural soil is the largest source of nitrous oxide (N₂O), a greenhouse gas. Soybean is an important leguminous crop worldwide. Soybean hosts symbiotic nitrogen-fixing soil bacteria (rhizobia) in root nodules. In soybean ecosystems, N₂O emissions often increase during decomposition of the root nodules. Our previous study showed that N₂O reductase can be used to mitigate N₂O emission from soybean fields during nodule decomposition by inoculation with nosZ++ strains [mutants with increased N₂O reductase (N₂OR) activity] of Bradyrhizobium diazoefficiens. Here, we show that N₂O emission can be reduced at the field scale by inoculation with a mixed culture of indigenous nosZ+ strains of B. diazoefficiens USDA110 group isolated from Japanese agricultural fields. Our results also suggested that nodule nitrogen is the main source of N₂O production during nodule decomposition. Isolating nosZ+ strains from local soybean fields would be more applicable and feasible for many soybean-producing countries than generating mutants.

Methylobacterium inhabits the phyllosphere of a large number of plants. We herein report the results of comparative metagenome analyses on methylobacterial communities of soybean plants grown in an experimental field in Tohoku University (Kashimadai, Miyagi, Japan). Methylobacterium was identified as the most dominant genus (33%) among bacteria inhabiting soybean stems. We classified plant-derived Methylobacterium species into Groups I, II, and III based on 16S rRNA gene sequences, and found that Group I members (phylogenetically close to M. extorquens) were dominant in soybean-associated Methylobacterium. By comparing 29 genomes, we found that all Group I members possessed a complete set of genes for the N-methylglutamate pathway for methylamine utilization, and genes for urea degradation (urea carboxylase, urea amidolyase, and conventional urease). Only Group I members and soybean methylobacterial isolates grew in a culture supplemented with methylamine as the sole carbon source. They utilized urea or allantoin (a urea-related compound in legumes) as the sole nitrogen source; however, group III also utilized these compounds. The utilization of allantoin may be crucial in soybean-bacterial interactions because allantoin is a transported form of fixed nitrogen in legume plants. Soybean-derived Group I strain AMS5 colonized the model legume Lotus japonicus well. A comparison among the 29 genomes of plant-derived and other strains suggested that several candidate genes are involved in plant colonization such as csgG (curli fimbriae). Genes for the N-methylglutamate pathway and curli fimbriae were more abundant in soybean microbiomes than in rice microbiomes in the field. Based on these results, we discuss the lifestyle of Methylobacterium in the legume phyllosphere.

The relationships between biogeochemical processes and microbial functions in rice (Oryza sativa) paddies have been the focus of a large number of studies. A mechanistic understanding of methane-nitrogen (CH4-N) cycle interactions is a key unresolved issue in research on rice paddies. This minireview is an opinion paper for highlighting the mechanisms underlying the interactions between biogeochemical processes and plant-associated microbes based on recent metagenomic, metaproteomic, and isotope analyses. A rice symbiotic gene, relevant to rhizobial nodulation and mycorrhization in plants, likely accommodates diazotrophic methanotrophs or the associated bacterial community in root tissues under low-N fertilizer management, which may permit rice plants to acquire N via N2 fixation. The amount of N fixed in rice roots was previously estimated to be approximately 12% of plant N based on measurements of (15)N natural abundance in a paddy field experiment. Community analyses also indicate that methanotroph populations in rice roots are susceptible to environmental conditions such as the microclimate of rice paddies. Therefore, CH4 oxidation by methanotrophs is a driving force in shaping bacterial communities in rice roots grown in CH4-rich environments. Based on these findings, we propose a hypothesis with unanswered questions to describe the interplay between rice plants, root microbiomes, and their biogeochemical functions (CH4 oxidation and N2 fixation).