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Abstract

The diversity of methanotrophic bacteria associated with roots of
submerged rice plants was assessed using cultivation-independent
techniques. The research focused mainly on the retrieval of pmoA, which
encodes the alpha subunit of the particulate methane monooxygenase. A
novel methanotroph-specific community-profiling method was established
using the terminal restriction fragment length polymorphism (T-RFLP)
technique. The T-RFLP profiles clearly revealed a more complex
root-associated methanotrophic community than did banding patterns
obtained by pmoA-based denaturing gradient gel electrophoresis. The
comparison of pmoA-based T-RFLP profiles obtained from rice roots and
bulk soil of Hooded rice microcosms suggested that there was a
substantially higher abundance of type I methanotrophs on rice roots
than in the bulk soil. These were affiliated to the genera Methylomonas,
Methylobacter, Methylococcus, and to a novel type I methanotroph
sublineage. By contrast, type II methanotrophs of the
Methylocystis-Methylosinus group could be detected with high relative
signal intensity in both soil and root compartments. Phylogenetic
treeing analyses and a set of substrate-diagnostic amino acid residues
provided evidence that a novel pmoA lineage was detected. This branched
distinctly from all currently known methanotrophs. To examine whether
the retrieval of pmoA provided a complete view of root-associated
methanotroph diversity, we also assessed the diversity detectable by
recovery of genes coding for subunits of soluble methane monooxygenase
(mmoX) and methanol dehydrogenase (mxaF). In addition, both 16S rRNA and
16S ribosomal DNA (rDNA) were retrieved using a PCR primer set specific
to type I methanotrophs. The overall methanotroph diversity detected by
recovery of mmoX, mxaF, and 16S rRNA and 16S rDNA corresponded well to
the diversity detectable by retrieval of pmoA.