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Applied and Environmental Microbiology, October 2007, p. 6519-6525, Vol. 73, No. 20
0099-2240/07/$08.00+0     doi:10.1128/AEM.01405-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Cultivation-Independent Analysis of Fungal Genotypes in Soil by Using Simple Sequence Repeat Markers{triangledown}

Kaspar Schwarzenbach, Franco Widmer, and Jürg Enkerli*

Molecular Ecology, Agroscope Reckenholz-Tänikon Research Station ART, Reckenholzstrasse 191, 8046 Zürich, Switzerland

Received 25 June 2007/ Accepted 12 August 2007

Cultivation-independent analyses of fungi are used for community profiling as well as identification of specific strains in environmental samples. The objective of the present study was to adapt genotyping based on simple sequence repeat (SSR) marker detection for use in cultivation-independent monitoring of fungal species or strains in bulk soil DNA. As a model system, a fungal biocontrol agent (BCA) based on Beauveria brongniartii, for which six SSR markers have been developed, was used. Species specificity of SSR detection was verified with 15 fungal species. Real-time PCR was used to adjust for different detection sensitivities of the six SSR markers as well as for different template quantities. The limit for reliable detection per PCR assay was below 2 pg target DNA, corresponding to an estimated 45 genome copies of B. brongniartii. The cultivation-independent approach was compared to cultivation-dependent SSR analysis with soil samples from a B. brongniartii BCA-treated field plot. Results of the cultivation-independent method were consistent with cultivation-dependent genotyping and allowed for unambiguous identification and differentiation of the applied as well as indigenous strains in the samples. Due to the larger quantities of soil used for cultivation-dependent analysis, its sensitivity was higher, but cultivation-independent SSR genotyping was much faster. Therefore, cultivation-independent monitoring of B. brongniartii based on multiple SSR markers represents a rapid and strain-specific approach. This strategy may also be applicable to other fungal species or strains for which SSR markers have been developed.

* Corresponding author. Mailing address: Molecular Ecology, Agroscope Reckenholz-Tänikon Research Station ART, Reckenholzstrasse 191, 8046 Zürich, Switzerland. Phone: 41 (0)44 377 72 06. Fax: 41 (0)44 377 72 01. E-mail: juerg.enkerli{at}art.admin.ch

{triangledown} Published ahead of print on 24 August 2007.

Applied and Environmental Microbiology, October 2007, p. 6519-6525, Vol. 73, No. 20
0099-2240/07/$08.00+0     doi:10.1128/AEM.01405-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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