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Applied and Environmental Microbiology, October 2001, p. 4479-4487, Vol. 67, No. 10
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.10.4479-4487.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Characterization of Bacterial and Fungal Soil Communities by Automated Ribosomal Intergenic Spacer Analysis Fingerprints: Biological and Methodological Variability

L. Ranjard,1 F. Poly,1 J.-C. Lata,1 C. Mougel,1,dagger J. Thioulouse,2 and S. Nazaret1,*

Laboratoire d'Ecologie Microbienne, UMR CNRS 5557,1 and Laboratoire de Biométrie, UMR CNRS 5558,2 Université Claude Bernard Lyon I, F-69622 Villeurbanne Cedex, France

Received 26 March 2001/Accepted 11 July 2001

Automated rRNA intergenic spacer analysis (ARISA) was used to characterise bacterial (B-ARISA) and fungal (F-ARISA) communities from different soil types. The 16S-23S intergenic spacer region from the bacterial rRNA operon was amplified from total soil community DNA for B-ARISA. Similarly, the two internal transcribed spacers and the 5.8S rRNA gene (ITS1-5.8S-ITS2) from the fungal rRNA operon were amplified from total soil community DNA for F-ARISA. Universal fluorescence-labeled primers were used for the PCRs, and fragments of between 200 and 1,200 bp were resolved on denaturing polyacrylamide gels by use of an automated sequencer with laser detection. Methodological (DNA extraction and PCR amplification) and biological (inter- and 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 technique and is a robust method for discriminating between microbial communities. To evaluate the potential biases in community description provided by ARISA, we also examined databases on length distribution of ribosomal intergenic spacers among bacteria (L. Ranjard, E. Brothier, and S. Nazaret, Appl. Environ. Microbiol. 66:5334-5339, 2000) and fungi.

* Corresponding author. Mailing address: UMR-CNRS 5557---Ecologie Microbienne, Université Claude Bernard Lyon I, Bât. 741, 4ème étage, 43 Bd. du 11 November 1918, F-69622 Villeurbanne Cedex, France. Phone: 33(4)72431324. Fax: 33(4)72431223. E-mail: nazaret{at}biomserv.univ-lyonl.fr.

dagger Present address: School of Biology, Georgia Institute of Technology, Atlanta, GA 30332-0230.

Applied and Environmental Microbiology, October 2001, p. 4479-4487, Vol. 67, No. 10
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.10.4479-4487.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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