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J Microbiol Methods. 2004 Nov;59(2):223-31.

RNA fingerprinting--a new method to screen for differences in plant litter degrading microbial communities.

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  • 1GSF-National Research Center for Environment and Health, Institute of Soil Ecology, PO Box 1129, Neuherberg D-85764, Germany.

Abstract

Microbial activities are essential for the nutrient turnover processes in soil and play an important role in the degradation of complex organic material, for example, plant leaf litter. However, very little is known about the microorganisms and their genes involved during the course of leaf litter decomposition. In the present study, we describe the non-radioactive application of RNA arbitrarily primed-PCR (RAP-PCR) protocol in combination with the classic litter bag technique to investigate the metabolic profiles of microbial community involved in leaf litter degradation after 2 and 8 weeks of degradation in four different soil sites, without using selective primer systems for PCR. Due to the significantly reduced target sites for PCR primers, compared to the published papers about RAP fingerprinting of more complex microbial communities based on DNA analysis (only transcripts from microbes on the litter material were analysed), the patterns of parallel samples were highly reproducible (>95%). Shifts in microbial community structure and function were observed during the course of degradation. Each litter sample had its unique metabolic profile and both soil effects and litter quality effects were evident. RAP-PCR products were also cloned to generate libraries. Clone libraries were screened by restriction fragment length polymorphism (RFLP) and representative samples sequenced to identify the inserts. Both mRNA and rRNA transcripts were obtained confirming the presence of mRNA in total RNA preparations. Hence, the described protocol is a good screening method to find similarities or differences in the structure and function of microbial communities involved in litter degradation, which may be the basis for more detailed studies by cloning and sequencing approaches.

PMID:
15369858
DOI:
10.1016/j.mimet.2004.07.005
[PubMed - indexed for MEDLINE]
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