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18

Detection of low numbers of Phakopsora pachyrhizi spores by quantitative real-time PCR

Presenter: T. A. Steinlage

All authors and affiliations: T. A. STEINLAGE (1), M. R. Miles (2), S. A. Isard (3), J. Marois (4), and G. L. Hartman (1,2). (1) Department of Crop Sciences, National Soybean Research Center, University of Illinois, Urbana, IL 61801; (2) USDA-ARS, National Soybean Research Center, University of Illinois, Urbana, IL 61801; (3) Department of Plant Pathology, Buckhout Lab, Penn State University, University Park, PA 16802; and (4) Department of Plant Pathology, North Florida Research and Education Center, University of Florida, Quincy, FL 32351

One important aspect of an early detection and monitoring system for soybean rust is the reliability of detection of low spore numbers from traps. Microscopic examination of slides is time-consuming and may provide inaccurate results due to look-alike spores (spores of a similar size, morphology, and color). PCR-based technologies offer the opportunity for rapid, definitive confirmation of suspect spores. Groups of spores (0, 1, 4, 8, 16, and 100) were removed from Vaseline-coated slides with a scalpel, and DNA was extracted by the FastDNA protocol. Detection was performed by quantitative real-time PCR. When 100 or 16 spores were extracted, detection was achieved with every sample. At lower levels of 8, 4, and 1 spores extracted, detection was achieved in 75, 88, and 38% of the samples, respectively. No false positives occurred in the negative controls. During the 2006 field season, this technology was applied to spore traps in Florida. More than 800 samples were processed, with 44 slide traps and 17 rainwater traps yielding positive signals for Phakopsora pachyrhizi. This technology shows promise as a method for confirming the identification of suspect spores and may aid in future early warning systems. Additional experimentation is ongoing to improve the accuracy of detection when spore numbers are low.

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