title: 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.
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.
authors.affiliation: Institute for Environmental Sciences, University of Koblenz-Landau, Landau, Germany.|Department of Aquatic Sciences and Assessment, Swedish University of Agricultural Sciences, Uppsala, Sweden.
authors.forename: Alexander
authors.initials: A
authors.lastname: Feckler
authors.affiliation: Institute for Environmental Sciences, University of Koblenz-Landau, Landau, Germany.
authors.forename: Anne
authors.initials: A
authors.lastname: Schrimpf
authors.affiliation: Institute for Environmental Sciences, University of Koblenz-Landau, Landau, Germany.|Department of Aquatic Sciences and Assessment, Swedish University of Agricultural Sciences, Uppsala, Sweden.
authors.forename: Mirco
authors.initials: M
authors.lastname: Bundschuh
authors.affiliation: Department of Biology, Mt. Allison University, Sackville, Canada.
authors.forename: Felix
authors.initials: F
authors.lastname: Bärlocher
authors.affiliation: Institute for Environmental Sciences, University of Koblenz-Landau, Landau, Germany.
authors.forename: Patrick
authors.initials: P
authors.lastname: Baudy
authors.affiliation: Laboratoire Interdisciplinaire des Environnements Continentaux, UMR CNRS 7360, Université de Lorraine, Metz, France.|Department of Experimental Limnology, Leibniz-Institute of Freshwater Ecology and Inland Fisheries (IGB), Stechlin, Germany.|MARE-Marine and Environmental Sciences Centre, Department of Life Sciences, University of Coimbra, Coimbra, Portugal.
authors.forename: Julien
authors.initials: J
authors.lastname: Cornut
authors.affiliation: Institute for Environmental Sciences, University of Koblenz-Landau, Landau, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Schulz
mesh_terms: D001203:Ascomycota / Q000235:genetics*; D005800:Genes, Fungal; D057230:Limit of Detection; D018515:Plant Leaves / Q000382:microbiology*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*