Upscaled CTAB-Based DNA Extraction and Real-Time PCR Assays for Fusarium culmorum and F. graminearum DNA in Plant Material with Reduced Sampling Error
Open Access
- 24 November 2008
- journal article
- research article
- Published by MDPI AG in International Journal of Molecular Sciences
- Vol. 9 (11), 2306-2321
- https://doi.org/10.3390/ijms9112306
Abstract
Fusarium graminearum Schwabe (Gibberella zeae Schwein. Petch.) and F. culmorum W.G. Smith are major mycotoxin producers in small-grain cereals afflicted with Fusarium head blight (FHB). Real-time PCR (qPCR) is the method of choice for species-specific, quantitative estimation of fungal biomass in plant tissue. We demonstrated that increasing the amount of plant material used for DNA extraction to 0.5-1.0 g considerably reduced sampling error and improved the reproducibility of DNA yield. The costs of DNA extraction at different scales and with different methods (commercial kits versus cetyltrimethylammonium bromide-based protocol) and qPCR systems (doubly labeled hybridization probes versus SYBR Green) were compared. A cost-effective protocol for the quantification of F. graminearum and F. culmorum DNA in wheat grain and maize stalk debris based on DNA extraction from 0.5-1.0 g material and real-time PCR with SYBR Green fluorescence detection was developed.Keywords
This publication has 36 references indexed in Scilit:
- Real-time PCR detection and quantification ofFusarium poae, F. graminearum, F. sporotrichioidesandF. langsethiaein cereal grains in Finland and RussiaArchives of Phytopathology and Plant Protection, 2008
- Strategies for managing Fusarium head blight and deoxynivalenol accumulation in wheatInternational Journal of Food Microbiology, 2007
- Global molecular surveillance reveals novel Fusarium head blight species and trichothecene toxin diversityFungal Genetics and Biology, 2007
- Quantification of Fusarium graminearum in infected wheat by species specific real-time PCR applying a TaqMan ProbeJournal of Microbiological Methods, 2004
- MANAGEMENT AND RESISTANCE IN WHEAT AND BARLEY TO FUSARIUM HEAD BLIGHTAnnual Review of Phytopathology, 2004
- PCR-based detection and quantification of mycotoxigenic fungiMycological Research, 2002
- Simultaneous One-Tube Quantification of Host and Pathogen DNA with Real-Time Polymerase Chain ReactionPhytopathology®, 2002
- Quantification of Trichothecene-Producing Fusarium Species in Harvested Grain by Competitive PCR To Determine Efficacies of Fungicides against Fusarium Head Blight of Winter WheatApplied and Environmental Microbiology, 2001
- Polymerase Chain Reaction-Based Assays for Species-Specific Detection ofFusarium culmorum, F. graminearum,andF. avenaceumPhytopathology®, 1996
- Fusarium ear blight (scab) in small grain cereals—a reviewPlant Pathology, 1995