To demonstrate the extent of phylogenetic diversity of diazotrophic bacteria associated with rice roots, we characterized phylogenetically 23 nifH gene sequences obtained by PCR amplification of mixed organism DNA extracted directly from rice roots without culturing the organisms. The analyses document the presence of eight novel NifH types, which appear to be a variety of significant components of the diazotrophic community, dominated mainly by proteobacteria.The method developed by Pace et al. to determine species diversity and composition, using rRNA (or ribosomal DNA) isolated directly from nature has opened a window into the world of unculturable bacteria, although this method has the disadvantage that organisms whose genes have been isolated by the method cannot be studied for any other trait (11,16). This report extends the method by applying it to a functionally important gene, nifH, to determine the importance and diversity of nitrogen-fixing bacteria associated with rice roots. The nitrogenase iron protein gene nifH is one of the oldest existing and functioning genes in the history of gene evolution, and the outline of the NifH tree is reported to be largely consistent with the 16S rRNA phylogeny (21,22). These features prompted us to study the genetic diversity of N 2 -fixing bacteria by the molecular evolutionary analysis of nifH sequences amplified directly from rice root DNA, because the rice root DNA contains not only plant DNA but also microbial DNA in the roots. In the rice root zone, N 2 fixation is associated with the activity of N 2 -fixing heterotrophic bacteria (4,20). It has been reported that a large percentage of the total aerobic heterotrophic population in the root is diazotrophic (1, 10, 17). However, studies on the rhizospheric N 2 -fixing microflora have until now suffered from the use of selective media for counts and isolations, because it is widely believed that only a small percentage of natural prokaryotes may actually be culturable (18,19). Here we report a study of N 2 -fixing bacterial diversity by analysis of nifH gene sequences without a cultivation technique.DNA extraction. Rice, Oryza sativa L. cv. nihonnbare, was raised under flooded conditions in the Kyushu University Farm. Rice plants taken at the heading stage in September were dug out from a wetland rice field, and the roots were washed to remove the attached soil. The washed roots were cut into segments, frozen with liquid nitrogen, and ground to a fine powder in a mortar and pestle. The fine powder was suspended in extraction buffer (100 mM Tris, 100 mM EDTA, 250 mM NaCl, 100 g of proteinase K per ml) supplemented with Sarkosyl (1% final concentration) and lysed by incubation at 55ЊC for 1 h. Treatment of the lysate with RNase A was followed by chloroform extraction and isopropanol precipitation. Crude DNA was purified by phenol extraction, chloroform extraction, and isopropanol precipitation.PCR amplification of nifH genes. The primers for PCR amplification were chosen by careful inspection of the 37 published ni...

To understand the host specificity of rhizobia and the relationship between the evolution of Sym plasmids and that of host plants, we determined partial nodC sequences of 10 representative rhizobium strains and then constructed an evolutionary tree for the deduced amino acid sequences with four published sequences. These coding sequences yield a phylogenetic tree similar to that for leghemoglobin of host plants, suggesting that the evolution of common nodulation genes may be linked to host legume evolution and speciation.

The rhizosphere of wetland rice has significant N2-fixing activity. It has been suggested that N2 fixation in the rice root zone is associated with the activity of various N2-fixing heterotrophic bacteria that inhabit the rice rhizosphere. Because of the generic diversity, many different isolation media and conditions are required to count and isolate these bacteria. In an attempt to overcome any bias from culture-dependent methods we amplified nifD segments from crude rice root DNA by the polymerase chain reaction. The nifD fragments were then cloned into a pT7 Blue T-vector to construct a nifD library. Sixteen cloned nifD genes chosen at random from the library were sequenced. A comparison with published sequences indicated the presence of seven novel groups of NifD proteins, which implies the existence of at least seven components in the diazotrophic community of rice roots, dominated mainly by proteobacteria. We also observed genetic variability within the clusters, which suggests the coexistence of many closely related bacterial lineages. However, we did not find Azospirillum-like nifD clones, although many reports indicated the widespread presence of Azospirillum spp. Therefore, it remains to be clarified whether Azospirillum species are the widespread N2-fixing bacteria in rice roots.