Quantification of fungal abundance on cultural heritage using real time PCR targeting the β-actin gene

Abstract

The traditional methodology used for the identification of microbes colonizing our cultural heritage was the application of cultivation methods and/or microscopy. This approach has many advantages, as living microorganisms may be obtained for physiological investigations. In addition, these techniques allow the quantitative and qualitative assessment of the investigated environment. Quantitative analyses are done by plate count and the determination of abundance by the colony forming unit (CFU). Nevertheless, these techniques have many drawbacks that lead to an underestimation of the cell numbers and do not provide a comprehensive overview of the composition of the inhabiting microbiota. In the last decades, several molecular techniques have been developed enabling many advantages over the cultivation approach. Mainly PCR-based, fingerprinting techniques allow a qualitative detection and identification of the microbiota. In this study, we developed a real time PCR method as a simple, rapid and reliable tool to detect and quantify fungal abundance using the β-actin gene, which is known to appear as a single-copy gene in fungi. To this end, five different indoor thermal insulation materials applied for historical buildings that were previously tested for their bio-susceptibility against various fungi were subjected to qPCR analyses. The obtained results were compared with those obtained from a previous study investigating the bio-susceptibility of the insulation materials using classical cultivation experiments. Both results correlated well, revealing that Perlite plaster was the most suitable insulation material, showing the lowest fungal CFU and qPCR values. In contrast, insulations made of wood showed to be not recommendable from the microbiological point of view. In addition, the potential of qPCR was tested in other materials of cultural heritage, as old parchments, showing to be a suitable method for measuring fungal abundance in these delicate materials.

The cell-wall-localised BETA-XYLOSIDASE 4 contributes to immunity of Arabidopsis against Botrytis cinerea

“…The mpk3 mutant was used as a control with enhanced susceptibility (Galletti et al, 2011). An additional method was used to quantify the disease susceptibility to B. cinerea , which involved spraying the plants with B. cinerea conidiospore suspension and quantifying the fungal β-ACTIN genomic DNA using qPCR (Ettenauer et al, 2014). Fungal DNA was quantified immediately after spraying (0 dpi) and 72 h after spray inoculation (3 dpi).…”

“…For fungal DNA quantification, fungal DNA was extracted using plant/fungi DNA isolation kit (Norgen Biotek Corp) following the manufacturer’s protocol. The fungal β -ACTIN genomic DNA was quantified by qPCR (Ettenauer et al, 2014) and primers used are listed in supplemental table S1.…”

Identification of Phytotoxic and Phytopathogenic Fungi on Grains and Wheat Seedlings

“…Primers for the β-actin protein encoding gene, ACT 512-F and ACT 783-R, developed by Carbone and Kohn [9], have already been successfully applied to the development of a valuable methodology for assessing the degree of fungal contamination of objects in cultural heritage or historical sites [26].…”

Identification and selection of normalization controls for quantitative transcript analysis in Blumeria graminis

“…In addition, TUBB, H3 and MGLL were previously identified as stable genes during the B. graminis-barley interaction (Both et al, 2005). The three genes TUBA, TUBB and ACTB have all been used in other fungal species, including Melampsora larici-populina (Hacquard et al, 2011), Fusarium graminearum (Brown et al, 2011), Magnaporthe oryzae (Kim et al, 2009), Cladosporium cladosporioides, Aspergillus niger and Penicillium chrysogenum (Ettenauer et al, 2014).…”