Open AccessCultivation-Independent Analysis of Fungal Genotypes in Soil by Using Simple Sequence Repeat Markers
Author(s) -
Kaspar Schwarzenbach,
Franco Widmer,
J. Enkerli
Publication year - 2007
Publication title -
applied and environmental microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.552
H-Index - 324
eISSN - 1070-6291
pISSN - 0099-2240
DOI - 10.1128/aem.01405-07
Subject(s) - biology , genotyping , beauveria , microsatellite , genotype , dna profiling , genetic marker , genetics , dna , biological pest control , botany , gene , allele , entomopathogenic fungi
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 onBeauveria 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 ofB. brongniartii . The cultivation-independent approach was compared to cultivation-dependent SSR analysis with soil samples from aB. 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 ofB. 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.