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Characterization of myxomycete communities in soil by reverse transcription polymerase chain reaction (RT-PCR)-based method

Kamono, Akiko, Matsumoto, Jun, Kojima, Hisaya, Fukui, Manabu
Soil biology & biochemistry 2009 v.41 no.6 pp. 1324-1330
soil fungi, reverse transcriptase polymerase chain reaction, RNA, Myxomycetes, fruiting bodies, soil-plant interactions, optimization, gel electrophoresis, seasons, community structure, Japan
Myxomycetes are amoeboid organisms, and their life cycle includes a stage in which they produce fruiting bodies. Extensive field collections of their fruiting bodies have been conducted in various terrestrial ecosystems. However, little is known about the ecology of myxomycetes in their natural habitat, especially during their trophic stages. To study the presence and distribution of myxomycetes such as those belonging to Didymiaceae and Physaraceae in soil, we used a polymerase chain reaction (PCR)-based method. First, optimization of the reverse transcription (RT)-PCR conditions was performed with the RNA extracted from soil samples. A semi-nested RT-PCR approach was used to improve the specificity of detection. We used newly and previously designed primers in the first round of amplification, which was followed by a second round with the previously described primer pair for PCR-denaturing gradient gel electrophoresis. Next, this method and nucleotide sequence analyses were applied to study several soil samples collected from five study sites. While no marked dynamic seasonal change was detected in the community composition of myxomycetes belonging to Didymiaceae and Physaraceae in the soil samples collected from the single site, their composition significantly differed between different sites. This is the first study that demonstrates the presence of myxomycetes in the soil and characterizes their community composition by RNA-based molecular analysis.