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Biological nitrogen fixation (BNF) by plants and its bacterial associations represent an important natural system for capturing atmospheric dinitrogen (N 2 ) 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 N 2 fixation in non-leguminous plants. Identification of the involved N 2 -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 N 2 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 N 2 . In paddy rice fields, methane molecules generated under anoxia are oxidized by xylem aerenchyma-transported oxygen with the simultaneous fixation of N 2 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.

Molecular Analyses of the Distribution and Function of Diazotrophic Rhizobia and Methanotrophs in the Tissues and Rhizosphere of Non-Leguminous Plants