Validation and Application of a PCR Primer Set to Quantify Fungal Communities in the Soil Environment by Real-Time Quantitative PCR

Abstract

Fungi constitute an important group in soil biological diversity and functioning. However, characterization and knowledge of fungal communities is hampered because few primer sets are available to quantify fungal abundance by real-time quantitative PCR (real-time Q-PCR). The aim in this study was to quantify fungal abundance in soils by incorporating, into a real-time Q-PCR using the SYBRGreen® method, a primer set already used to study the genetic structure of soil fungal communities. To satisfy the real-time Q-PCR requirements to enhance the accuracy and reproducibility of the detection technique, this study focused on the 18S rRNA gene conserved regions. These regions are little affected by length polymorphism and may provide sufficiently small targets, a crucial criterion for enhancing accuracy and reproducibility of the detection technique. An in silico analysis of 33 primer sets targeting the 18S rRNA gene was performed to select the primer set with the best potential for real-time Q-PCR: short amplicon length; good fungal specificity and coverage. The best consensus between specificity, coverage and amplicon length among the 33 sets tested was the primer set FR1 / FF390. This in silico analysis of the specificity of FR1 / FF390 also provided additional information to the previously published analysis on this primer set. The specificity of the primer set FR1 / FF390 for Fungi was validated in vitro by cloning - sequencing the amplicons obtained from a real time Q-PCR assay performed on five independent soil samples. This assay was also used to evaluate the sensitivity and reproducibility of the method. Finally, fungal abundance in samples from 24 soils with contrasting physico-chemical and environmental characteristics was examined and ranked to determine the importance of soil texture, organic carbon content, C∶N ratio and land use in determining fungal abundance in soils.

Plant functional diversity enhances associations of soil fungal diversity with vegetation and soil in the restoration of semiarid sandy grassland

“…Concurrently, many studies have also suggested that soil fungal diversity is strongly dependent on soil properties (Prevost‐Boure et al. 2011; Siciliano et al. 2014; LeBlanc et al.…”

Chemical and microbiological changes in Norway spruce deadwood during the early stage of decomposition as a function of exposure in an alpine setting

“…To build the standard curves we used as templates the purified PCR products of known concentrations of the following pure cultures: Fusarium solani (DSMZ 10696), fungi, and Azospirillum irakense (DSMZ 11586), nifH gene. The primer pairs were FF390/FR1 (fungi; Prévost-Bouré et al 2011) and nifHf/nifHr (nifH gene; Töwe et al 2010). Stock concentration (gene copies µL −1 ) was determined via PicoGreen measurement and was freshly prepared for each run.…”

Genetic characterization of <i>Salmonella</i> and <i>Shigella</i> spp. isolates recovered from water and riverbed sediment of the Apies River, South Africa

“…Genetic approaches also provide additional information such as pathogenicity, especially if the genes targeted are those associated with the disease-causing abilities of the organism (Pathak et al, 2012). As a result, qPCR has been used to enumerate bacteria in different sample types, including food (Elizaquível et al, 2012), soil (Prévost-Bouré et al, 2011), wastewater (Sidhu et al, 2013 and marine sediments (Luna et al, 2012;Schippers et al, 2012). Despite these numerous applications, the use of qPCR in riverbed sediment studies is still in its early stages.…”