Quantitative real-time PCR as a promising tool for the detection and quantification of leaf-associated fungal species - A proof-of-concept using Alatospora pulchella

Alexander Feckler; Anne Schrimpf; Mirco Bundschuh; Felix Bärlocher; Patrick Baudy; Julien Cornut; Ralf Schulz

doi:10.1371/journal.pone.0174634

Quantitative real-time PCR as a promising tool for the detection and quantification of leaf-associated fungal species - A proof-of-concept using Alatospora pulchella

PLoS One. 2017 Apr 6;12(4):e0174634. doi: 10.1371/journal.pone.0174634. eCollection 2017.

Authors

Alexander Feckler^{1

2}, Anne Schrimpf¹, Mirco Bundschuh^{1

2}, Felix Bärlocher³, Patrick Baudy¹, Julien Cornut^{4

5

6}, Ralf Schulz¹

Affiliations

¹ Institute for Environmental Sciences, University of Koblenz-Landau, Landau, Germany.
² Department of Aquatic Sciences and Assessment, Swedish University of Agricultural Sciences, Uppsala, Sweden.
³ Department of Biology, Mt. Allison University, Sackville, Canada.
⁴ Laboratoire Interdisciplinaire des Environnements Continentaux, UMR CNRS 7360, Université de Lorraine, Metz, France.
⁵ Department of Experimental Limnology, Leibniz-Institute of Freshwater Ecology and Inland Fisheries (IGB), Stechlin, Germany.
⁶ MARE-Marine and Environmental Sciences Centre, Department of Life Sciences, University of Coimbra, Coimbra, Portugal.

Abstract

Traditional methods to identify aquatic hyphomycetes rely on the morphology of released conidia, which can lead to misidentifications or underestimates of species richness due to convergent morphological evolution and the presence of non-sporulating mycelia. Molecular methods allow fungal identification irrespective of the presence of conidia or their morphology. As a proof-of-concept, we established a quantitative real-time polymerase chain reaction (qPCR) assay to accurately quantify the amount of DNA as a proxy for the biomass of an aquatic hyphomycete species (Alatospora pulchella). Our study showed discrimination even among genetically closely-related species, with a high sensitivity and a reliable quantification down to 9.9 fg DNA (3 PCR forming units; LoD) and 155.0 fg DNA (47 PCR forming units; LoQ), respectively. The assay's specificity was validated for environmental samples that harboured diverse microbial communities and likely contained PCR-inhibiting substances. This makes qPCR a promising tool to gain deeper insights into the ecological roles of aquatic hyphomycetes and other microorganisms.

Publication types

Validation Study

MeSH terms

Ascomycota / genetics*
Genes, Fungal*
Limit of Detection
Plant Leaves / microbiology*
Real-Time Polymerase Chain Reaction / methods*

Grants and funding

This work was supported by the German Research Foundation (DFG) (grant number SCHU 2271/14-1), the Carl Tryggers Foundation, Stockholm (grant number CTS 13:72), and the Fix Stiftung, Landau. Financial support by the Portuguese Foundation for Science and Technology to Julien Cornut (SFRH/BPD/108779/2015) is gratefully acknowledged. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.