Title: Recovery of Phakopsora pachyrhizi urediniospores from Passive Spore Trap Slides and Extraction of Their DNA for Quantitative PCR Authors
Submitted to: American Phytopathology Society
Publication Type: Abstract Only
Publication Acceptance Date: July 13, 2009
Publication Date: June 1, 2010
Citation: Haudenshield, J.S., Chaudhary, P., Hartman, G.L. 2010. Recovery of Phakopsora pachyrhizi urediniospores from Passive Spore Trap Slides and Extraction of Their DNA for Quantitative PCR [abstract]. North-Central Division of the American Phytopathological Society Regional Meeting, June 21-23, 2009, Ames, Iowa. 100:S185. Technical Abstract: Enumeration of rust spores from passive spore traps utilizing white petrolatum-coated slides by traditional microscopic evaluation can represent a serious challenge. Many fungal spores look alike, and clear visualization on the adhesive can be obscured by particulate debris or nonuniformities within the adhesive layer; reports will commonly describe only the number of “rust-like” spores. Molecular methods of P. pachyrhizi detection, utilizing both standard PCR and quantitative PCR (qPCR), have been available for several years, but extraction of fungal DNA from petrolatum-embedded spores remained difficult. We now demonstrate the utility of a novel method for recovering the petrolatum layer carrying trapped spores from slides using biodegradable foam strips, with subsequent DNA extraction to yield material suitable for quantification by qPCR. This method permits even single spores of P. pachyrhizi to be recovered and detected. False-negative calls were minimized by using a multiplexed exogenous control; no false-positives were observed. This method was successfully employed to assess spore loads in passive traps located at sentinel plots in the USA during the 2008 soybean growing season.