We examined the nitrogenase reductase (nifH) genes of endophytic diazotrophic bacteria expressed in field-grown sweet potatoes (Ipomoea batatas L.) by reverse transcription (RT)-PCR. Gene fragments corresponding to nifH were amplified from mRNA obtained from the stems and storage roots of field-grown sweet potatoes several months after planting. Sequence analysis revealed that these clones were homologous to the nifH sequences of Bradyrhizobium, Pelomonas, and Bacillus sp. in the DNA database. Investigation of the nifH genes amplified from the genomic DNA extracted from these sweet potatoes also showed high similarity to various α-proteobacteria including Bradyrhizobium, β-proteobacteria, and cyanobacteria. These results suggest that bradyrhizobia colonize and express nifH genes not only in the root nodules of leguminous plants but also in sweet potatoes as diazotrophic endophytes.

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N 2 fixation systems in the nonleguminous crops and bacteria associations have been intensively studied over the last 50 years. Their structure and regulation have been investigated to explore the enhancement of N acquisition in these ecosystems leading to crop-growth with minimum chemical fertilizers. Several lines of important evidence have been accumulated indicating that the magnitudes of associative (nonsymbiotic) N 2 fixation in sugarcane (Saccharum spp.), sweet potato (Ipomoea batatas L.), and paddy rice (Oryza sativa L.) are agronomically significant. In these three crops, unique bacterial N 2fixation systems may function in addition to the low-level activity (due to the competition in carbon/ energy use) of the commonly occurring rhizosphere-associated system by free-living bacteria such as Beijerinckia, Azotobacter, and Klebsiella. Active expressions of the dinitrogenase reductase-encoded gene (nifH) phylogenetically similar to those of Bradyrhizobium spp. and Azorhizobium sp. were abundantly found in the N 2 -fixing sugarcane stems, sweet potato stems, and storage tubers. These rhizobia microaerobically fix N 2 in the carbon compounds-rich apoplasts. Gluconacetobacter diazotrophicus and Herbaspirillum spp. were previously isolated from inside the sugarcane stems, as the candidates of endophytic N 2 fixers. However, the current molecular and physiological investigations suggest that their major role is production of phytohormonal substances. In paddy rice fields, methane is produced from organic compounds in anoxia and oxidized by contacting with oxygen gas. An active N 2 -fixation by methane-oxidizing methanotrophs such as Methylosinus sp. takes place in the root tissues (aerenchyma) and also in the surface soil. This methanotrophic N 2 -fixation supports the sustainability of soil fertility although the N 2 -fixation and soil fertility are affected by chemical fertilizers. Finally, we discuss the ecological implications of the newly identified rhizobia and methanotroph systems in the N nutrition in nonlegumes and N reservation in field environments. ARTICLE HISTORY

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.