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Applied and Environmental Microbiology, January 2004, p. 240-247, Vol. 70, No. 1
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.1.240-247.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

New Molecular Screening Tools for Analysis of Free-Living Diazotrophs in Soil

Helmut Bürgmann,1* Franco Widmer,2 William Von Sigler,1 and Josef Zeyer1

Soil Biology, Institute of Terrestrial Ecology, Swiss Federal Institute of Technology Zurich, 8952 Schlieren,1 Swiss Federal Research Station for Agroecology and Agriculture (FAL-Reckenholz), 8046 Zurich, Switzerland2

Received 14 March 2003/ Accepted 29 September 2003

Free-living nitrogen-fixing prokaryotes (diazotrophs) are ubiquitous in soil and are phylogenetically and physiologically highly diverse. Molecular methods based on universal PCR detection of the nifH marker gene have been successfully applied to describe diazotroph populations in the environment. However, the use of highly degenerate primers and low-stringency amplification conditions render these methods prone to amplification bias, while less degenerate primer sets will not amplify all nifH genes. We have developed a fixed-primer-site approach with six PCR protocols using less degenerate to nondegenerate primer sets that all amplify the same nifH fragment as a previously published PCR protocol for universal amplification. These protocols target different groups of diazotrophs and allowed for direct comparison of the PCR products by use of restriction fragment length polymorphism fingerprinting. The new protocols were optimized on DNA from 14 reference strains and were subsequently tested with bulk DNA extracts from six soils. These analyses revealed that the new PCR primer sets amplified nifH sequences that were not detected by the universal primer set. Furthermore, they were better suited to distinguish between diazotroph populations in the different soils. Because the novel primer sets were not specific for monophyletic groups of diazotrophs, they do not serve as an identification tool; however, they proved powerful as fingerprinting tools for subsets of soil diazotroph communities.

* Corresponding author. Mailing address: Soil Biology, Institute of Terrestrial Ecology, Swiss Federal Institute of Technology Zurich, 8952 Schlieren, Switzerland. Phone: 41-1-633 6046. Fax: 41-1-633 1122. E-mail: helmut.buergmann{at}cnv.ethz.ch.

Applied and Environmental Microbiology, January 2004, p. 240-247, Vol. 70, No. 1
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.1.240-247.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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