Cereal Disease Laboratory : Detection of Phakopsora pachyrhizi DNA in rain using qPCR and a portable rain collector

CW Barnes^{1, 2}, LJ Szabo^{1, 2}, JL Johnson¹, K-P. Nguyen¹, CM Floyd², JE Kurle²¹USDA-ARS Cereal Disease Laboratory, St. Paul, MN, ²University of Minnesota, St. Paul, MN

INTRODUCTION

A real-time PCR (qPCR) assay was previously developed for detection of Phakopsora pachyrhizi (causal agent of Asian Soybean Rust-ASR) in filtered rain using precipitation collectors from the National Atmospheric Deposition Program (NADP). NADP collectors are widely distributed, but are deployed in specific locations. Therefore, we developed portable and inexpensive “JB” collectors and distributed thirty across Minnesota near soybean fields and sentinel plots during the 2005 growing season. Filters were mailed to the Cereal Disease Laboratory and assayed for P. pachyrhizi.

OBJECTIVES

To develop a portable and inexpensive rain collector.
To adapt the qPCR assay used with NADP to the portable collector system.
To test filtered rain samples for P. pachyrhizi spores in soybean fields and sentinel plots across Minnesota.

METHODS

1. Collect spores

Fig. A. A JB collector near a soybean field in southern Minnesota.

2. Remove spores from filters by sonication

3. DNA extraction. The PowerSoil™ DNA extraction kit was used.

Fig. B. An example of a filter after one week in the field.

4. Nested real-time PCR assay

Fig.C. 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 qPCR posisitves

Filters were exposed to wet and dry deposition for about 1 week.
Filters testing positive for P. pachyrhizi by the qPCR assay were scored as 1 and divided by the number of days the filter was on the collector.

Distribution of Soybean in Minnesota

Fig. D. Distribution of JB collectors. Yellow counties indicate where soybean is grown.

RESULTS

Fig. E. Score values were calculated by taking the reciprocal of the number of days each positive filter was exposed and summed over a given week. Daily rain values were summed for counties with JB collectors. See geographic representation of week 7/20 though 7/27 below.

Fig. F. Minnesota counties with precipitation from 7-20-05 to 7-27-05. Also corresponds to counties with detectable P. pachyrhizi DNA. No other samples were positive.

Fig. G. Correlation between frequency of qPCR positives and latitude. (See Fig. H.)

Fig. H. Score values were calculated by taking the reciprocal of the number of days each positive filter was exposed and summed for a given county over the length of the study. Each county was plotted by its latitude.

SUMMARY

1. First detection of P. pachyrhizi in Minnesota was in mid June

2. Positive samples were found every week, with increased frequency in late July through August

3. Positive samples corresponded with rain events indicating long distance transport of P. pachyrhizi spores in rain

4. Frequency of detection was inversely proportional with latitude

5. Overall, frequency of detection was 22% of samples collected

ACKNOWLEDGEMENTS

Funding was provided by the USDA-ARS, Minnnesota Soybean Research and Promotion Council, and the Minnesota Agricultural Rapid Response fund. We’d also like to thank John Butler and Yibai Li for graphical assistance.