Public Security S&T Symposium 2009

1. CRTI-04-0045RDDevelopment of collections and reference / DNA databases and detection systems to counter bioterrorism against agriculture and forestry Public Security S&T Symposium 2009

2. Presentation outline • Relevance - Why Fungal Plant Pathogens? • Objectives and Summary of Achievements • Recent Technical Progress and Results • Benefits to Canada • Conclusion

3. 80% of plant pathogens are fungi – consistent with this list

4. § 331.3 List of biological agents and toxins. (a) The biological agents and toxins listed in this section have been determined to have the potential to pose a severe threat to plant health or to plant products. Liberobacter africanus, Liberobacter asiaticus Peronosclerospora philippinensis Phakopsora pachyrhizi Plum pox potyvirus Ralstonia solanacearum, race 3, biovar 2 Sclerophthora rayssiae var. zeae Synchytrium endobioticum Xanthomonas oryzae pv. oryzicola Xylella fastidiosa (citrus variegated chlorosis strain)‏

5. Challenges for Plant Diseases • There are many more plant than animal hosts. Each plant species has as many pathogens as human does. This project covered a range of plant pathogenic and mycotoxigenic fungi that could be used against plants and humans (in the case of mycotoxins). • Canadian research in this area is necessary in order to monitor high risk fungi and thereby ensure the safety of the food supply and our continued ability to export agricultural and forestry products. • Our successes in this project were dependent on our ability to adapt to emerging priorities. The project demonstrated the immediate benefits of proactive R&D efforts into plant bio-terrorism threats.

6. Agriculture and Agriculture et Agri-Food Canada Agroalimentaire Canada Ressources naturelles Natural Resources Canada Canada Keith Seifert Sarah Hambleton Scott Redhead Carolyn Babcock John Bissett Stéphan Brière Marie-Josée Côté Richard Hamelin The team leaders Team Leaders André Lévesque Project Manager Christopher Lewis Deputy Project Manager and Bioinformatics

7. Objectives and Summary of achievements • Genetic resource collections and databases • Updated of the Canadian phytopathogen/host literature database • Acquired target organisms and related species (21/23 species) • Identification of hypervariable genes for strain typing • Completed for organisms with available annotated genome sequences • Explored alternative approaches for organisms without whole genome sequences • Microsatellite flanking regions & EST libraries • Build up DNA sequence database • Database completed • Close to 32,000 sequence reactions, greatly exceeding the project requirements. • Detection tools for high risk plant pathogens • Q-PCR primers and probes designed for 19 species • More than one assay developed for most species • DNA array developed and preliminary validation completed

8. Recent Technical Progress and Results • Completion of the qPCR assays for remaining targets. • Validation of assays using environmental samples. • Validation of transferred assays at CFIA. • Development of an oligonucleotide array to allow parallel detection of targets.

9. Recent Technical Progress and Results- Rusts • Stem rust of wheat (P. graminis) & testing of Microsatellite Flanking Region markers: • one locus differentiated isolates collected in Africa from isolates from other geographic regions. • Stripe rust of wheat (P. striiformis) and crown rust of oats (P. coronata) are species complexes comprising multiple sub-specific groups infecting a broad range of wild and cultivated grasses/cereals. • The assays developed in this project are diagnostic for the groups that infect crop hosts. Puccinia striiformis

10. Real-Time PCR Assay — Fusarium sporotrichioides Standard curve for efficiency calculation derived from dilution series Fluorescence signals of samples • TaqMan assay specific for F. sporotrichioides • Probe: FAM – CGC TTT TGC CCT TCC CAC ACA TCC AT- BHQ • Assay is negative for F. langsethiae and other closely related species • Validated on 150 strains from 5 continents and many different hosts

11. RealTime-PCR Assay — Fusarium graminearum TaqMan probe: TET - TCG TTG AGC CTT CTG AGT ACT TTG GGT TGT - BHQ MAT locus • TaqMan assay specific for F. graminearum s.str. • Validation on 250 geo-graphically diverse strains

12. 1 2 3 4 5 6 7 8 9 10 11 A ST1 Pcor1-ITS1 Pcor2-ITS1 Pcor-Btub Pcor1-Btub Gabi1-Btub Gabi2-Btub Gabi3-Btub Pstr1-ITS1 Pstr2-ITS1 ST1 B Univ-lo58S-199B Pcor2-Btub Pcor3-Btub Pcor4-Btub Pcor5-Btub Gabi4-Btub Gabi5-Btub Gabi1-Btub-lna Pstr3-ITS1 Pstr-Btub Pstr1-Btub C Universal 5.8S-3 Pcor6-Btub Pcor7-Btub Pcor8-Btub Pcor3-RPB2 Gabi2-Btub-lna Gabi3-Btub-lna Gabi4-Btub-lna Pstr-RPB2 Pstr1-RPB2 Pstr1-CoxI D lmbd600-U220 Pcor4-RPB2 Pcor5-RPB2 Pgra-ITS1 Pgra1-ITS1 Pgra2-ITS1 Tind-Mit Gabi5-Btub-lna Pstr2-CoxI Pstr3-CoxI Pstr4-CoxI E lmbd600-L400 Send-ITS Pram-ITS Pgra3-ITS1 Pgra4-ITS1 Pgra-Btub Cpol-HMG Pinf-ITS1 Pinf-ITS2 Pinf-Nadh1 Pinf-Nadh1-lna F lmbd800-U354 Send-18S Pram-Btub Pgra1-Btub Pgra2-Btub Pgra-RPB2 Ppac-ITS1 Paln-ITS1 Paln-ITS2 Paln-CoxII Paln-CoxII-lna G lmbd800-L524 Send-IGS Pram-Elic Pgra1-RPB2 Pgra3-RPB2 Pgra-CoxI Fgra-MAT Pque-ITS1 Pque-ITS2 Pque-CoxI Pque-CoxI-lna Recent Technical Progress and Results- DNA Array Hybridization • Individual Taqman probes were converted to array oligos. • The primers from the Taqman assays were assayed for compatibility in multiplex amplifications.

13. Recent Technical Progress Diagnostic Array Setup - DIG detection system Exposure to film or camera DIG-dUTP Dig-labeled PCR Probe 5’ Amino-modified Oligo bound to membrane

14. Recent Technical Progress Hybridization (Phytophthora quercina) Cross- hybridization Controls Controls Cross- hybridization Target

15. Benefits to Canada – Response to Crises,Protocol Transfers to First Responders • The Q-PCR for P. ramorum was implemented at CFIA and helped keep Canada free of this Quarantine organism. • The first detection in Canadian history of a plant pathogen (Phakopsora pachyrhizi) in air and rain samples before a diseased plant was found. • Record outbreaks of Fusarium in corn production and the need for testing for mycotoxin producing fungi. • Re-occurrence of potato wart in PEI and need for the assay being developed on this project.

16. Benefits to Canada Phytophthora ramorum diagnostics at CFIA to prevent introduction • 58,000 putative P. ramorum samples processed at CFIA since April 1, 2007. • Of these 18,000 by the CRTI species specific realtime PCR assay –317 positives detected. • Infested area cleaned up and Canada is still considered free of P. ramorum for trade. NRCan award CFIA award Stéphan Brière sampling infested nursery in BC

17. Benefits to Canada - Asian Soybean Rust “real-time” Monitoring JB collector Burkhard air sampler Work by Dr. Sarah Hambleton in collaboration with Ontario/Quebec Provincial Ministries & Ontario/Manitoba Grower Organizations Spore collectors to monitor rust through DNA-based technology Loda rainfall collector

18. Benefits to Canada – Asian Soybean Rust Monitoring • In 2007, the first Canadian molecular detection of soybean rust spores occurred for rainfall and air samples from collectors deployed at 12 sites in Canada (ON, MB, SK) • In October 2007, the first Canadian infected soybean plant was found in southwestern Ontario • The spore monitoring program continued in 2008 & 2009. In 2008, positive detections from rainfall and air samples indicated widespread deposition across a broad sampling area in late June/early July) showing that long-range transport of spores is possible early in the growing season, at the most critical time for disease development in Canada. Strong collaboration with USDA on this project. USDA models showing the projected movement of spores into Ontario from Arkansas, Missouri, and Oklahoma in September. Example shown is for September 28 to 30, 2007 (from http://www.sbrusa.net)

19. Benefits to Canada – Fusarium Head Blight assays • 2006 - the biggest epidemic in history of Fusarium graminearum on corn in Ontario. • Bioethanol production sometimes uses mycotoxin contaminated feedstock, which must be monitored for worker and environmental safety • Our TaqMan assay is being tested for regulatory use by Canadian Grain Commission (CGC) • Direct quantification of fungal DNA extracted from wheat in collaboration with CGC

20. Benefits to Canada – Potato Wart assays • This pathogen is on the US Agriculture Bioterrorism Act Select Agent List. • We developed three Q-PCR assays. Protocol for preparation of clean spores for validation assays Homogenization ... centrifugation ... before ... after enzyme

21. Benefits to Canada – Protocols from Canada adopted by trading partners • USDA-APHIS signed an MTA for validation material and assays for their own testing of potato wart with CRTI protocols. • USDA-APHIS officially adopted the CRTI elicitin assay for P. ramorum testing throughout US. • The European Commission just launched the Quarantine Barcode of Life (QBOL) initiative for plant pathogens. A member of our team is a scientific advisor. http://www.aphis.usda.gov/plant_health/plant_pest_info/

22. Benefits to Canada – Preparedness beyond CRTI-04-0045RD wheat in Kenya Ug99 wheat stem rust – A new global threat 1 June 2009

23. Benefits to Canada – Preparedness beyond CRTI-04-0045RD • The Asian Soybean rust network is now being used to test assays for three other rust diseases developed in this project - stem rust and stripe rust of wheat, and crown rust of oats. • Special funding under AAFC Growing Forward initiative was approved for Ug99, the new threat. • CRTI project has provided Canada with critical data for this research. international group for wheat resistance rating to Ug99

24. Benefits to Canada – Highly Qualified Personnel CRTI Post-CRTI Deputy Project Manager;Bioinformaticist Bioinformaticist for Microbiota study. Leading Pyrosequencing approach. Christopher Lewis

25. Benefits to Canada – Highly Qualified Personnel CRTI Post-CRTI Post-Doc;Fusarium(Mycotoxin) Canadian Grain Commission; AAFC/Sloan Foundation project on indoor air quality. Dr. Tom Graefenham

26. Benefits to Canada – Highly Qualified Personnel CRTI Post-CRTI Post-Doc;Rusts Post-Doc AAFC; Ug99 project pending final approval of funding. Dr. Miao (Mindy) Liu

27. Benefits to Canada – Highly Qualified Personnel CRTI Post-CRTI Post-Doc;Zoosporic fungi Research Scientist, Microbiology, Dept. Fisheries and Ocean, Nanaimo. Dr. Cathryn Abbott

28. Benefits to Canada – Highly Qualified Personnel CRTI Post-CRTI Ph.D. candidate;Phytophthora ramorum Post-Doc, Plant Pathology, USDA - Agriculture Research Services, Salinas, CA. Dr. Guillaume Bilodeau

29. Benefits to Canada – Highly Qualified Personnel CRTI Post-CRTI Technician AAFC EG04;Molecular Biology Technician EG05 for AAFC Microbiota study. Pyrosequencing approach. Julie Chapados

30. Benefits to Canada – Highly Qualified Personnel CRTI Post-CRTI Technician CFIA EG04;Molecular Biology Technician CFIA EG04; Molecular Biologyindeterminate. Marie-Ève Auclair

31. Benefits to Canada – Highly Qualified Personnel CRTI Post-CRTI Technician EG04;Rusts Biologist, CFIA Plant Diagnostic Lab. Ray Tropiano

32. CONCLUSIONS • Even after the completion of CRTI-04-0045RD, fruitful national collaboration continues among AAFC, NRCan/CFS and CFIA in Plant Biosecurity and a new level of international collaboration has been achieved. • Technology transfers are completed to respond to emergencies and unexpected high priorities. • An extensive collection of high risk pathogens and their relatives has been developed and is being shared among Canadian partners. • Databases for literature, pathogens, DNA and sequences were developed and shared. • Several detection assays were validated and some are already implemented for routine testing by end users.