Analysis of rain samples for Phakopsora pachyrhizi

Transcription

1 Analysis of rain samples for Phakopsora pachyrhizi Les J. Szabo Charlie Barnes USDA ARS Cereal Disease Lab Dept. of Plant Pathology University of Minnesota St. Paul, Minnesota Van Bowersox NADP Illinois State Water Survey Champaign, IL Jim Kurle Dept. of Plant Pathology University of Minnesota St. Paul, Minnesota

2 Introduction Spores of rust fungi Airborne Travel long distances Puccinia graminis (wheat stem rust)

3 Puccinia Pathway

4 Wheat stem rust June 8, 2004 July 6, 2004

5 Rain Collectors

6 Rain collectors: NADP Active, collects only wet deposition Approximately 14 diameter 0.45 micron filter Weekly collections

7 NADP/NTN sites

8 Rain collectors: JB Passive, collects both wet and dry deposition 17 diameter funnel Whatman filter holder 8 micron filter Filter holder changed weekly

9 Kittson Marshall Rosea u Pennington Red Lake Polk Norman Mahnowmen Minnesota Agricultural Regions North West West Central South West Central South Central South East East Clay Becker Wilkin Otter Tail Wadena Pipestone Brown MurrayCottonwood Watonwan Rock Nobles Jackson Martin Crow Wing Blue Earth Aitkin Le Sueur Rice Goodhue Wabasha Waseca Dodge Steele Faribault Freebor n Carlton Pine Todd Mille Grant Morrison Lacs Douglas Kanabe Traver Big se Stevens c Pope Stearns Isant Stone i Chisago Swift Tail Kandiyohi Anoka Lac Wright Washington Meeker Qui Chippewa Hennepi Ramsey Parle Renville McLeo Carver n Yellow Medicine Dakota d Scott Lincoln Lyon Redwood Sibley Nicollet Mower Olmste d Winon a Fillmore Houston Site Locations

10 Real-time PCR assay

11 Real-time PCR assay Based on nuclear rdna ITS region Single step assay Two step Nested assay

12 Real-time PCR assay Ssu ITS-1 5.8S ITS-2 One step Rust specific primers Ph. pachyrhizi specific TaqMan probe

13 Real-time PCR assay Ssu ITS-1 5.8S ITS-2 Nested-1 (Two step) Rust specific primer pair (set 1) Ph. pachyrhizi specific primer pair (set 2) Ph. pachyrhizi specific TaqMan probe

14 Real-time PCR assay Ssu ITS-1 5.8S ITS-2 Nested-2 (Two step) Ph. pachyrhizi specific primer pair (set 1) Rust specific primer pair (set 2) Ph. pachyrhizi specific TaqMan probe

15 Real-time PCR assay Single step assay Ph. pachyrhizi specific Detection limit (approximately 200 fg DNA) Nested assay Ph. pachyrhizi specific Detection limit (< 1 fg DNA) Nested-2 assay (Ph. pachyrhizi specific primer in PCR-1) was insensitive to high levels of other rust spores

16 Real-time PCR assay: Results

17 Real-time PCR: NADP 122 sites May 10 - October 8, 2005 > 3,000 samples Processed >1,600 samples May 10 - Aug positive samples Amplification products of positive samples were analyzed on agarose gels Amplification products of selected positive samples were cloned and sequenced

18 Real-time PCR: qpcr scale 1 = trace () Fl > 10 SD above background Usually no visible amplification product on gel 2 = low () Fl >15 SD above background PCR amplification product visible on gel and correct size 3 = moderate () Fl > 25 SD above background PCR amplification product visible on gel and correct size

19 NADP sites: Positive samples 04 May 10 - August 30 = 3 = 1 = 2 qpcr scale

20 Real-time PCR assay: JB Samples from JB collectors are dirtier than samples from NADP collectors

21 Real-time PCR assay: JB Samples from JB collectors are dirtier than samples from NADP collectors DNA samples from JB samples show PCR inhibition using DNA spike experiments

22 Summary Real-time PCR assay was developed for testing rain samples for Ph. pachyrhizi DNA Rain samples from across the US soybean growing area were tested 85 samples tested positive for Ph. pachyrhizi DNA Positive samples were found in most of the regions test, including the Midwest and Northeast

23 Summary (cont.) These results indicate that Ph. pachyrhizi spores are easily transported via atmosphere and can travel long distances Infection and disease development are complex processes and presence of spores (inoculum) is only one factor

24 Summary (cont.) Work that needs to be done Calibrate Real-time PCR assay to actual amount of spores in sample Finish processing remaining samples Correlate PCR assay results with field data Place NADP collectors at selected sentinel plots across the southeast Refine protocol to accommodate dirty filters from JB collectors

25 Thanks USDA ARS CDL Charlie Barnes Jerry Johnson Kim-Phuong Nguyen Dept. Plant Pathology, UMN Jim Kurle Crystal Floyd Amy Holm NADP, ISWS Van Bowersox Karen Harlin Funding USDA ARS Minnesota Soybean Research & Promotion Council United Soybean Board Minnesota Agricultural Rapid Response Fund

26 Processing filters Filters cut in half 1/2 - DNA analysis 1/2 - Archived Particulate matter removed by sonication Bead basher used to disrupt spores DNA extraction (Omni kit)

27 Positive samples: May 04 qpcr scale = 1 = 2 = 3

28 Positive samples: June 04 qpcr scale = 1 = 2 = 3

29 Positive samples: July 04 qpcr scale = 1 = 2 = 3

30 Positive samples: August 04 qpcr scale = 1 = 2 = 3