Submitted to: Journal of Sugarbeet Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 1, 2002
Publication Date: January 1, 2003
Citation: LARTEY, R.T., WEILAND, J.J., CAESAR, T., BUCKLIN COMISKEY, S.A. A PCR protocol for rapid detection of Cercospora beticola in infected sugar beet tissues. JOURNAL OF SUGARBEET RESEARCH. 2003. V. 40 P. 1-10. Interpretive Summary: A PCR based protocol for rapid detection of Cercospora beticola in infected sugar beet leaves was developed. The protocol uses actin gene sequence and ITS region based primers to amplify target segments without DRA extraction and purification. Amplified action segment was confirmed by DNA sequencing. The protocol should enable rapid detection of C. beticola in infected sugar beet tissues and secondary hosts such as weeds.
Technical Abstract: Leaf spot, caused by Cercospora beticola. Sacc. is the most important foliar disease of sugarbeet (Beta vulgaris L), yet the progression of infection from soil to diseased leaves remains incompletely understood. A method for sensitive detection of C. beticola on disease-free plants could be used to determine how early in the growing season sugarbeet tissues are colonized by the fungus and to what extent asymptomatic weeds and non-beet crops harbor the fungus. We present an Extract-N-Amp Plant PCR Kit (Sigma)-based protocol for rapid detection and identification of C. beticola in plant tissues. Leaf disks from field-sampled diseased tissues or disease-free greenhouse plants were homogenized and diluted with manufacturer-provided extraction and dilution solutions. Without further DNA purification, aliquots of the homogenate were added to PCR reactions and subjected to amplification using the Cercospora actin gene specific and ITS region based primers. Fragment sizes of the amplified products correlated with the size of that amplified from DNA extracted from C. beticola cultures. Sequences alignment of the amplified products confirmed them to be that of C. beticola. The system will enable rapid detection and identification of C. beticola in asymptomatic and diseased sugarbeet, in alternate hosts and soil debris that harbor the fungus.