|Haudenshield, James - UNIV. OF ILLINOIS|
|Steinlage, Todd - UNIV OF ILLINOIS|
Submitted to: APSnet Resource Center
Publication Type: Abstract Only
Publication Acceptance Date: October 30, 2007
Publication Date: December 12, 2007
Citation: Haudenshield, J.S., Steinlage, T.A., Hartman, G.L. 2007. Quantification and Single-Spore Detection of Phakopsora pachyrhizi [abstract]. APSnet Resource Center. 2007 National Soybean Rust Symposium, December 12-14, 2007. Available: http://www.apsnet.org/online/SBR/. Technical Abstract: The microscopic identification and quantification of Phakopsora pachyrhizi spores from environmental samples, spore traps, and laboratory specimens can represent a challenge. Such reports, especially from passive spore traps, commonly describe the number of “rust-like” spores; for other forensic samples, visualization is impossible because of the accompanying milieu. Molecular methods of P. pachyrhizi detection, utilizing both standard PCR and quantitative PCR (Q-PCR), have been available for several years, and have proven useful in discriminating P. pachyrhizi from other rust fungi, and for quantifying DNA and the equivalent number of spores. We report here the validation of a combined method for DNA extraction and analysis by Q-PCR that reliably detects, identifies, and quantifies small numbers, and even single spores, of fresh P. pachyrhizi mechanically picked from an agar substrate. Seventy percent of single spores, 95% of spore pairs, and 100% of eight-spore samples were detected using this method. We compare the efficiency of this method for assaying fresh spores with that for heat-killed spores and for actively germinating spores picked from similar agar substrates, as well as for spores picked from white petrolatum-coated glass slides, as used in common spore traps.