Novel euryarchaeotal lineages detected on rice roots and in the anoxic bulk soil of flooded rice microcosms

Because excised, washed roots of rice (Oryza sativa) immediately produce CH 4 when they are incubated under anoxic conditions (P. Frenzel and U. Bosse, FEMS Microbiol. Ecol, 21:25-36, 1996), we employed a culture-independent m olecular approach to identify the methanogenic microbial community present on roots of rice plants. Archaeal small-subunit rRNA-encoding genes were am plified directly from total root DNA by PCR and then cloned. Thirty-two arc haeal rice root (ARR) gene clones were randomly selected, and the amplified primary structures of ca, 750 nucleotide sequence positions were compared. Only 10 of the environmental sequences were affiliated with known methanog ens; 5 were affiliated with Methanosarcina spp., and 5 were affiliated with Methanobacterium spp. The remaining 22 ARR gene clones formed four distinc t lineages (rice clusters I through IV) which were not closely related to a ny known cultured member of the Archaea, Rice clusters I and II formed dist inct clades within the phylogenetic radiation of the orders "Methanosarcina les" and Methanomicrobiales. Rice cluster I was novel, and rice cluster II was closely affiliated with environmental sequences obtained from bog peat in northern England. Rice cluster III occurred on the same branch as Thermo plasma acidophilum and marine group II but was only distantly related to th ese taxa, Rice cluster IV was a deep-branching crenarchaeotal assemblage th at was closely related to clone pGrfC26, an environmental sequence recovere d from a temperate marsh environment. The use of a domain-specific oligonuc leotide probe in a fluorescent in situ hybridization analysis revealed that viable members of the Archaea were present on the surfaces of rice roots. In addition, we describe a novel euryarchaeotal main line of descent, desig nated rice cluster V, which was detected in anoxic rice paddy soil. These r esults indicate that there is an astonishing richness of archaeal diversity present on rice roots and in the surrounding paddy soil.