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United States Department of Agriculture

Agricultural Research Service

Research Project: BIOLOGY, BIOLOGICAL CONTROL, AND MOLECULAR GENETICS OF ROOT DISEASES OF WHEAT, BARLEY AND BIOFUELS BRASSICAS

Location: Root Disease and Biological Control Research

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

Authors
item Poole, G -
item Ozdemir, F -
item Nydam, S -
item Schroeder, Kurtis
item Schroeder, Kurtis
item Paulitz, Timothy
item Nicol, J -
item Campbell, Kimberly

Submitted to: Phytopathology
Publication Type: Abstract Only
Publication Acceptance Date: May 15, 2009
Publication Date: June 2, 2009
Citation: Poole, G.J., Ozdemir, F., Nydam, S.D., Schroeder, K.L., Paulitz, T.C., Nicol, J.M., Campbell, K. 2009. Optimization of Real Time Quantitative PCR (Q-PCR) for Fusarium pseudograminearum and F. culmorum on wheat. Phytopathology 99: S103

Technical Abstract: 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-BIO© 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 Biomedicals© - FastDNA extraction kit)) and a Fast Prep speed of 5 for 45s. Addition of polyvinylpolypyrrolidone (PVPP) was necessary for adequate DNA extraction.

Last Modified: 10/1/2014
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