This data was presented as a poster at the 2006 Asian Soybean Rust Symposium in St. Louis, Missouri,November 29 through December 1, 2006

C.W. Barnes^{1, 2}, L.J. Szabo^{1, 2}, V.C. Bowersox³, K.S. Harlin³

¹CDL, USDA-ARS, St. Paul, MN; ²University of Minnesota, St. Paul, MN; ³NADP, Illinois State Water Survey, Champaign, IL.

OBJECTIVES

Our goal has been to monitor the movement of P. pachyrhizi spores in rain, in the eastern half of the US, using a real-time PCR assay and the National Atmospheric Deposition Program rain collectors. 

METHODS

1. Collect and filter rain

2. Removal of spores from filters by sonication

3. DNA extraction - The OmniPrep™ 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 primer (F), rust specific primers (R), and the P. pachyrhizi specific primer (Pp) developed by Frederick et al., 2002.  The TaqMan probe (Pp) is also specific for P. pachyrhizi.

5. Scoring real-time PCR positives

1. Filters were exposed to wet deposition for 1 week.
2. Filters testing positive for P. pachyrhizi by the qPCR assay were scored as 1 (Ct > 30) 2 (Ct = 25-29) 3 (Ct = 20-24) 4 (Ct < 20).

Fig. C. Schematic of scoring scale used in this study.  Scores are relative to an internal positive control.

6. Sequence a subset of amplicons

RESULTS

Contour maps showing numbers of NADP sites testing positive for P. pachyrhizi using the nest real-time PCR assay for 2006
	Number of positives

Fig. D. Weekly changes in positive rain samples is shown in the bar graph.

Geographical distribution of moderate to high relative scores (>2) in 2006
	Number of positives

Fig. E.  Temporal distribution of all relative scores in 2006. 

Fig. F. Distribution of amplicon genotypes of the ITS1 rDNA sequence of P. pachyrhizi found in rain collected May 9 - August 29, 2006.  Both polymorphisms are found near the 3’ end of ITS1, with the first letter designating a single nucleotide polymorphism 5 bases before the TA repeat. 

SUMMARY

• Positives were found in every state except North Dakota in 2006.
• Roughly 3 times as many samples tested positive for P. pachyrhizi in 2006 (270, 17%) compared to 2005 (84, 5%).
• Bimodal temporal pattern in spore deposition was similar in 2005 and 2006.
• Frequency of detection and spore load increased in Aug. 2006.
• Distribution of genotypes from rain samples suggests spores are transported long distances (over states) and that Region 2 may be at a crossroads for spore movement.

ACKNOWLEDGEMENTS

Funding was provided by the USDA-ARS and the United Soybean Board. We’d also like to thank Jacquie Koch, John Butler, Yibai Li, Brenda Riney (pictured), and John Rosnow for their technical assistance.