Detection of Methanotroph Diversity on Roots of Submerged Rice Plants by Molecular Retrieval of pmoA , mmoX , mxaF , and 16S rRNA and Ribosomal DNA, Including pmoA - Based Terminal Restriction Fragment Length Polymorphism Profiling

Abstract

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 α 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 flooded 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 .