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

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 (10 (9)) 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.

Suitability of Quantitative Real-Time PCR To Estimate the Biomass of Fungal Root Endophytes

“…3B and C). Similarly, hyphal length correlated positively with the number of ITS loci of Piloderma croceum grown in agar on microscope slides (36).…”

“…The method has been on May 13, 2018 by guest http://aem.asm.org/ used to estimate the biomass of plant pathogenic fungi in colonized plant tissues (8,9,32), EMF growing in artificial medium and in seedlings (26,36,40), and AMF in plant roots (9,12). However, the usefulness of qPCR and comparability with other methods to quantify fungal biomass in environmental samples has sometimes been controversial (12,26).…”

“…This was proposed to be due to the multinucleate nature of Glomeromycota. On the other hand, a correlation between qPCR and mycelial dry weight was demonstrated for the Swiss needle cast-causing parasite Phaeocryptopus gaeumannii (52) and between qPCR and hyphal length for the ectomycorrhizal fungus (EMF) Piloderma croceum (36). Partial correspondence between ergosterol assays and qPCR has been demonstrated in needles infected with P. gaeumannii (51), and a high correlation between qPCR and ergosterol was found in tissues colonized by the conifer root pathogen Heterobasidion annosum (25).…”

Impact of Endochitinase-Transformed White Spruce on Soil Fungal Biomass and Ectendomycorrhizal Symbiosis

“…Real-time PCR provides useful tools for studying and quantifying mycorrhizal fungus interactions and biomass (33,47,53,59). Unlike workers in previous studies, we did not use the ITS rRNA region to determine the number of Wilcoxina molecules.…”

Diversität der Ektomykorrhizen in verschieden artenreichen Laubbaumbeständen im Nationalpark Hainich (Thüringen)

“…Ein weiterer Vorteil ist, dass die ITS-Sequenz im Genom der Pilze sehr häufig vorkommt (Viaud et al 2000). Raidl et al (2005) fanden in unterschiedlichen Ektomykorrhizapilzen 120 -197 ITS-Kopien pro Zelle.…”