REAL-TIME PCR ASSAY TO QUANTIFY FUSARIUM GRAMINEARUM WILD-TYPE AND RECOMBINANT MUTANT DNA IN PLANT MATERIAL

Authors: Dyer, Rex; Kendra, David; Brown, Daren

Submitted to: Journal of Microbiological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/16/2006
Publication Date: 12/1/2006
Citation: Dyer, R.B., Kendra, D.F., Brown, D.W. 2006. Real-time PCR assay to quantify Fusarium graminearum wild-type and recombinant mutant DNA in plant material. Journal of Microbiological Methods. 67(2006):534-542.

Interpretive Summary: Fusarium Head Blight (FHB) or wheat scab is a devastating international disease caused primarily by the fungus Fusarium graminearum. When outbreaks of this disease occur, billions of dollars are lost due to poor grain quality and yield. During the disease process, the fungus can also create a toxin called deoxynivalenol (DON) which also significantly reduces grain value. DON is toxic to both plants and animals and ingestion by livestock can lead to a variety of affects like bleeding and anemia. In order to limit the negative affects of this fungus on wheat, we need to determine which genes are important to the production of the mycotoxin DON as well as the disease process or fungal/plant interactions. A common strategy to determine gene function is to create a strain of the fungus that has a mutant copy of a gene and then test if the lack of the gene affects toxin synthesis or the ability to cause disease. It is critical in these experiments to be able to make sure that any changes in disease are due to the specific strain being examined. We have developed a protocol based on the polymerase chain reaction assay to specifically detect and quantify F. graminearum and any mutant strains in contaminated plant material. We describe the specificity, sensitivity and accuracy of our protocol as applied to both pure fungal cultures and material from infected wheat.

Technical Abstract: Fusarium graminearum, the predominant causal agent of wheat head scab, is a major economic and food safety concern. In addition to a reduction in yield, infected wheat may also lead to seed contamination by the fungal generated toxin, deoxynivalenol (DON). DON is toxic to both plants and animals and is also a critical component of pathogenicity of wheat. Wheat contaminated with DON exacerbates the economic loss during grain marketing through added expenses for screening and testing. Given these concerns, it is imperative that we understand the pathway(s) for DON synthesis so that effective control strategies can be developed. Through gene knockout technology, genes and pathways suspected of being important in the pathogenesis of F. graminearum are being tested in the greenhouse and in the field. These studies depend on technology that allows the researcher to distinguish between disease caused by the parental strain and disease caused by the mutant, test strain. The recent advance of quantitative PCR has enabled the detection and quantification of fungi from field samples at the genus and species levels. Nicholson, et al. (Physiological and Molecular Plant Pathology, 53:17-37) has developed primers (Fg16F/R) to specifically detect and quantify F. graminearum from infected grain. In this report, we describe the development of a real-time quantitative PCR protocol using the Fg16F/R primers. In addition, we developed primers specific to our gene knockout technology. Given the documented specificity of the Fg16F/R primers and the widespread use of gene knockout technology, our protocol will be of general use to the plant pathology community for studying the pathobiology of F. graminearum.