Quantitative detection of agar-cultivated and rhizotron-grown Piloderma croceum Erikss. & Hjortst. by ITS1-based fluorescent PCR

Abstract

A real-time quantitative TaqMan-PCR was established for the absolute quantification of extramatrical hyphal biomass of the ectomycorrhizal fungus Piloderma croceum in pure cultures as well as in rhizotron samples with non-sterile peat substrate. After cloning and sequencing of internal transcribed spacer (ITS) sequences ITS1/ITS2 and the 5.8S rRNA gene from several fungi, including Tomentellopsis submollis, Paxillus involutus, and Cortinarius obtusus, species-specific primers and a dual-labelled fluorogenic probe were designed for Piloderma croceum. The dynamic range of the TaqMan assay spans seven orders of magnitude, producing an online-detectable fluorescence signal during the cycling run that is directly related to the starting number of ITS copies present. To test the confidence of the PCR-based quantification results, the hyphal length of Piloderma croceum was counted under the microscope to determine the recovery from two defined but different amounts of agar-cultivated mycelia. Inspection of the registered Ct values (defined as that cycle number at which a statistically significant increase in the reporter fluorescence can first be detected) in a 10-fold dilution series of template DNA represents a suitable and stringent quality control standard for exclusion of false PCR-based quantification results. The fast real-time PCR approach enables high throughput of samples, making this method well suited for quantitative analysis of ectomycorrhizal fungi in communities of natural and artificial ecosystems, so long as applicable DNA extraction protocols exist for different types of soil.