Optimization of Real Time Quantitative PCR (Q-PCR) for Fusarium pseudograminearum and F. culmorum on wheat

Fusarium crown rot of wheat is caused by a complex of Fusarium species, of which F. pseudograminearum and F. culmorum are the most important. Crown rot reduces wheat yields by an average of 9% in the Pacific Northwest. Traditional methods of species identification have included morphological characteristics of macroconidia. With the advent of Q-PCR techniques and the development of primers for F. pseudograminearum and F. culmorum, the potential exists for more accurate species identification and fungal DNA quantification from infected wheat stems. Primers developed in previous studies were evaluated for use in Q-PCR and DNA extraction kits were tested and optimized to accurately assess Fusarium species and DNA concentrations in wheat stem tissue. The 'OPT' primers (Shilling et al. 1996) for the amplification of F. culmorum and the 'FPG' primers (Williams et al. 2002) for the amplification of F. pseudograminearum yielded the most consistent results. The MO-BIOo Ultra Clean Soil Kit for DNA extraction produced the most consistent Q-PCR amplification from infected wheat tissue. The most optimal results were obtained by grinding with liquid nitrogen and soaking prior to bead beating (using a ceramic bead (MP Biomedicalso - FastDNA extraction kit)) and a Fast Prep speed of 5 for 45s. Addition of polyvinylpolypyrrolidone (PVPP) was necessary for adequate DNA extraction.