Plant Biol (Stuttg) 2005; 7(6): 713-717
DOI: 10.1055/s-2005-873003
Research Paper

Georg Thieme Verlag Stuttgart KG · New York

Calibration of Quantitative Real-Time Taqman PCR by Correlation with Hyphal Biomass and ITS Copies in Mycelia of Piloderma croceum

S. Raidl1 , R. Bonfigli1 , R. Agerer1
  • 1Department Biologie I und GeoBio-CenterLMU, Biodiversitätsforschung: Systematische Mykologie, Ludwig-Maximilians-Universität München, Menzinger Straße 67, 80638 München, Germany
Further Information

Publication History

Received: May 23, 2005

Accepted: October 25, 2005

Publication Date:
02 January 2006 (online)

Abstract

DNA-based quantification methods such as real-time TaqMan PCR allow a rapid and highly sensitive species-specific quantification of isolated fungal DNA material, but most quantification systems are only able to measure relative amounts of biomass or biomass changes during different treatments. In this experiment, an already established DNA quantification system for the ectomycorrhizal fungus Piloderma croceum, based on the ITS region of ribosomal DNA, was calibrated to absolute biomass to obtain a direct correlation between mycelial biomass and isolated ITS copies. Thin layers of sterile mycelia were cultured on slides. The mycelial biomass was calculated from measurements of the total hyphal length using image analysis, followed by determination of the mycelial volume, and multiplied by the specific weight of hyphae obtained from literature data. Using the very same mycelium, the number of ITS copies was quantified by TaqMan PCR. The mean value of 1047 (± 185) copies per mm hypha results in possible data for a direct conversion: one billion (109) ITS copies corresponded to 0.79 mg hyphal dry weight. For the ribosomal ITS multi-copy genes, the number of ITS copies could be calculated to approx. 152 (± 26) copies per dikaryotic cell. These conversion data now allow determination of the mycelial biomass of Piloderma croceum using real-time TaqMan PCR, a prerequisite for competition experiments with Piloderma croceum.

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S. Raidl

Department Biologie I und GeoBio-Center LMU
Biodiversitätsforschung: Systematische Mykologie
Ludwig-Maximilians-Universität München

Menzinger Straße 67

80638 München

Germany

Email: s.raidl@lrz.uni-muenchen.de

Guest Editor: R. Matyssek