|Detection of Puccinia graminis Spores in Rain Using a Real-time PCR Assay|
CW Barnes1, LJ Szabo1, 2, JL Johnson1, VC Bowersox3, SV Krupa2, DA Gay3, KS Harlin3
1USDA-ARS, St. Paul, MN, 2University of Minnesota, St. Paul, MN
3National Atmospheric Deposition Program,
Atmospheric transport and deposition constitutes the major mechanism for dispersal of certain rust fungi. Trap nurseries, currently used to follow seasonal movements of rusts, are labor intensive and reflective of past events. An early warning system for the arrival of spores is needed. Puccinia graminis was used as a model rust system to test whether real-time PCR can detect rust fungal spores in rain samples. The data were used to create back trajectory models to determine the source of inoculum.
1. Collect spores
2. Remove spores from filters
Fig. A. Puccinia graminis spores were spotted onto a filter, and visualized at 200X (left). The filter was sonicated for 1 minute at 60 oC (right).
3. DNA extraction. The OmniPrep™ DNA extraction kit was used.
4. Nested real-time PCR assay
Fig. B. Schematic of the real-time PCR assay. Arrows represent primers and the bar represents the TaqMan probe. Primers used were a general fungal primers (F), rust specific primers (R). The TaqMan probe (Pg) is specific for P. graminis.
5. Detection limits
Distribution of Wheat Along the Puccinia Pathway
Maps of stem rust progression and spore detection.
Back Trajectory Model
· Low levels of spores were detected 4 weeks prior to 1st observed infections in the field.
· Moderate levels of spores were detected 3 weeks prior to 1st observed infections in the field.
· High levels of spores were detected by mid June in Kansas and Missouri and may be from local inoculum sources.
Funding was provided by the USDA-ARS CRIS, Minnnesota Soybean Research and Promotion Council, and the Minnesota Agricultural Rapid Response fund. We’d also like to thank Jacki Morrison for graphical assistance.