Establishment and application of real-time PCR-based methods to study the epidemiology of Fusarium Head Blight

Abstract

Fusarium head blight (FHB) is a disease of cereal crops, which has a severe impact on wheat production worldwide. Apart from reducing the yield and impairing grain quality, FHB leads to contamination of grain with mycotoxins. The Fusarium species primarily involved in FHB are F. graminearum and F. culmorum, both producing deoxynivalenol (DON), the most important mycotoxin in wheat grain. Although the relative importance of F. graminearum and F. culmorum and their infection pathways has great impact on epidemiological questions, it has not be clarified in all details so far. For the differentiation of these pathogens, two real-time PCR methods were developed and applied, which allow a qualitative and quantitative detection of fungal biomass within host tissue. Samples for this study have been collected throughout Germany from 2003 to 2005. F. graminearum and F. culmorum were determined qualitatively in symptomatic wheat rachides with a duplex PCR assay enabling the identification of the causal agent of FHB in a single PCR reaction. The method is based on the analysis of melting curves of amplification products generated in a real-time PCR thermocycler. The quantitative detection of both fungi in complex plant material like wheat flour and plant debris was realized in separated real-time PCR reactions, applying SYBR Green I fluorescence detection. The specificity of both assays for F. graminearum and F. culmorum was confirmed. Suitable high-throughput DNA extraction methods were developed for all types of samples analyzed in this study, including wheat rachides, grain samples and plant debris. For quantitative PCR, an upscaled DNA extraction protocol with increased extraction efficiency was used to reduce the sampling error. In addition to the analysis with the quantitative PCR, the DON content of the grain samples was measured. The scope of the study was both the identification of the infecting agent in the wheat rachides and the determination of the relative importance of F. graminearum and F. culmorum in relation to the DON content of wheat samples to establish a risk assessment model for FHB. The results show that F. graminearum is the main causing agent of FHB and DON contamination in Germany. F. culmorum was sometimes detected in wheat rachides of 2003, but did not play a significant role in wheat grain samples 2003 to 2005. The collected data serve to adjust quantitative parameters of a computer-based decision support system aiming at the minimization of Fusarium infection and toxin contamination of grain in Germany, which is being developed by our collaborators.