Characterization of bacterial and fungal soil communities by automated ribosomal intergenic spacer analysis fingerprints: Biological and methodological variability

Automated rRNA intergenic spacer analysis (ARISA) was used to characterise bacterial (B-ARISA) and fungal (F-ARISA) communities from different soil ty pes. The 16S-23S intergenic spacer region from the bacterial rRNA operon wa s amplified from total soil community DNA for B-ARISA. Similarly, the two i nternal transcribed spacers and the 5.8S rRNA gene (ITS1-5.8S-ITS2) from th e fungal rRNA operon were amplified from total soil community DNA for F-ARI SA. Universal fluorescence-labeled primers were used for the PCRs, and frag ments of between 200 and 1,200 by were resolved on denaturing polyacrylamid e gels by use of an automated sequencer with laser detection. Methodologica l (DNA extraction and PCR amplification) and biological (interand intrasite ) variations were evaluated by comparing the number and intensity of peaks (bands) between electrophoregrams (profiles) and by multivariate analysis. Our results showed that ARISA is a high-resolution, highly reproducible tec hnique and is a robust method for discriminating between microbial communit ies. To evaluate the potential biases in community description provided by ARISA, we also examined databases on length distribution of ribosomal inter genic spacers among bacteria (L. Ranjard, E. Brothier, and S. Nazaret, Appl . Environ. Microbiol. 66:5334-5339, 2000) and fungi.