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.

Fungicides: An Overlooked Pesticide Class?

“…DNA-based methods such as metabarcoding and species- or group-specific quantitative real-time polymerase chain reaction provide tools for taxonomic identification and biomass quantification. 181 , 182 In addition, these methods offer new possibilities for solving major issues in stress and community ecology. Given their short generation times and small size, fungi are ideal model organisms to develop microcosm or field enclosure experiments to assess selection, adaptation, and their interaction with dispersal processes in (microbial) food webs.…”

Transcriptome Profiling of Elaeis guineensis Jacq. Under Heat Stress Condition

“…Primer was designed using Primer 3 online software (https://www.bioinformatics.nl/cgibin/primer3plus/primer3pl us.cgi). Gene expressions were analyzed in root and leaf samples using relative quantification [11]. Cq and quantity value of the sample were obtained automatically from the qPCR analysis software v2.0.6 7500, while Relative Quantitation (RQ) value was calculated using a formula based on Applied Biosystems guidance [12].…”

The Gibberellin Producer Fusarium fujikuroi: Methods and Technologies in the Current Toolkit

“…Besides, they can also distinguish the targeted sample in a complicated mixture. As an example, both ELISA and QPCR were used to quantify the biomass of filamentous fungi in infected plants (Brunner et al, 2012;Song et al, 2014;Feckler et al, 2017). However, when the cell sample is in a large quantity, such as the cells in fermentation, then the big dilution factor could confer significant error to these assays.…”