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

Presentation outline

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

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

§ 331.3 List of biological agents and toxins.(a) The biological agents and toxinslisted 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)

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.

The team leaders

Agriculture and Agriculture etAgri-Food Canada Agroalimentaire Canada

Ressources naturelles Natural ResourcesCanada Canada

Richard HamelinStéphan Brière

Marie-Josée Côté

Keith Seifert

Sarah Hambleton

Scott Redhead

Carolyn Babcock

John Bissett

Team

Leaders

André Lévesque Project ManagerChristopher Lewis Deputy Project Manager and Bioinformatics

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

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.

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

Real-Time PCR Assay — Fusarium sporotrichioides

• 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

Standard curve for efficiency calculation derived from dilution series Fluorescence signals of samples

RealTime-PCR Assay — Fusarium graminearum

TaqMan probe: TET - TCG TTG AGC CTT CTG AGT ACT TTG GGT TGT - BHQ

• TaqMan assay specific for F. graminearum s.str.

• Validation on 250 geo-graphically diverse strains

MAT locus

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.

Pque-CoxI-lna

Pque-CoxI

Pque-ITS2

Pque-ITS1

Fgra-MATPgra-CoxIPgra3-RPB2

Pgra1-RPB2

Pram-ElicSend-IGSlmbd800-

L524G

Paln-CoxII-lna

Paln-CoxII

Paln-ITS2Paln-ITS1Ppac-ITS1

Pgra-RPB2

Pgra2-Btub

Pgra1-Btub

Pram-Btub

Send-18Slmbd800-

U354F

Pinf-Nadh1-

lna

Pinf-Nadh1

Pinf-ITS2Pinf-ITS1Cpol-HMG

Pgra-BtubPgra4-ITS1

Pgra3-ITS1

Pram-ITSSend-ITSlmbd600-

L400E

Pstr4-CoxI

Pstr3-CoxI

Pstr2-CoxI

Gabi5-Btub-lna

Tind-MitPgra2-ITS1

Pgra1-ITS1

Pgra-ITS1Pcor5-RPB2

Pcor4-RPB2

lmbd600-U220D

Pstr1-CoxI

Pstr1-RPB2

Pstr-RPB2

Gabi4-Btub-lna

Gabi3-Btub-lna

Gabi2-Btub-lna

Pcor3-RPB2

Pcor8-Btub

Pcor7-Btub

Pcor6-Btub

Universal 5.8S-3

C

Pstr1-Btub

Pstr-BtubPstr3-ITS1

Gabi1-Btub-lna

Gabi5-Btub

Gabi4-Btub

Pcor5-Btub

Pcor4-Btub

Pcor3-Btub

Pcor2-Btub

Univ-lo58S-199BB

ST1Pstr2-ITS1

Pstr1-ITS1

Gabi3-Btub

Gabi2-Btub

Gabi1-Btub

Pcor1-Btub

Pcor-BtubPcor2-ITS1

Pcor1-ITS1

ST1A

1110987654321 

Recent Technical Progress

DIG-dUTP

Exposure to film or camera

Dig-labeled PCR Probe

5’ Amino-modified Oligo bound to membrane

Diagnostic Array Setup - DIG detection system

Recent Technical Progress

Hybridization (Phytophthora quercina)

Controls Controls

Target

Cross-hybridization

Cross-hybridization

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.

• 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.

Stéphan Brière sampling infested nursery in BC

CFIA awardNRCan award

Benefits to Canada Phytophthora ramorum diagnostics at CFIA

to prevent introduction

Benefits to Canada - Asian Soybean Rust “real-time” Monitoring

Spore collectors to monitor rust through DNA-based technology

JB collector

Loda rainfall collector

Burkhard air samplerWork by Dr. Sarah Hambleton in collaboration with Ontario/Quebec Provincial Ministries & Ontario/Manitoba Grower Organizations

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.

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)

Strong collaboration with USDA on this project.

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

Benefits to Canada – Potato Wart assays

This pathogen is on the US Agriculture Bioterrorism Act Select Agent List.We developed three Q-PCR assays.

Homogenization ... centrifugation ... before ... after enzyme

Protocol for preparation of clean spores for validation assays

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.

htt

p:/

/ww

w.a

ph

is.u

sda

.go

v/p

lan

t_h

ea

lth/p

lan

t_p

est

_in

fo/

1 June 2009

Benefits to Canada – Preparedness beyond CRTI-04-0045RD

Ug99 wheat stem rust – A new global threat wh

eat

in K

enya

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

Benefits to Canada – Highly Qualified Personnel

Deputy Project Manager;Bioinformaticist

Bioinformaticist for Microbiota study. Leading Pyrosequencing approach.

CRTI Post-CRTI

Christopher Lewis

Benefits to Canada – Highly Qualified Personnel

Post-Doc;Fusarium(Mycotoxin)

Canadian Grain Commission; AAFC/Sloan Foundation project on indoor air quality.

CRTI Post-CRTI

Dr. Tom Graefenham

Benefits to Canada – Highly Qualified Personnel

Post-Doc;Rusts

Post-Doc AAFC;Ug99 project pending final

approval of funding.

CRTI Post-CRTI

Dr. Miao (Mindy) Liu

Benefits to Canada – Highly Qualified Personnel

Post-Doc;Zoosporic fungi

Research Scientist, Microbiology, Dept. Fisheries and Ocean, Nanaimo.

CRTI Post-CRTI

Dr. Cathryn Abbott

Benefits to Canada – Highly Qualified Personnel

Ph.D. candidate;Phytophthora ramorum

Post-Doc, Plant Pathology, USDA - Agriculture Research Services, Salinas, CA.

CRTI Post-CRTI

Dr. Guillaume Bilodeau

Benefits to Canada – Highly Qualified Personnel

Technician AAFC EG04;Molecular Biology

Technician EG05 for AAFC Microbiota study. Pyrosequencing approach.

CRTI Post-CRTI

Julie Chapados

Benefits to Canada – Highly Qualified Personnel

Technician CFIA EG04;Molecular Biology

Technician CFIA EG04; Molecular Biologyindeterminate.

CRTI Post-CRTI

Marie-Ève Auclair

Benefits to Canada – Highly Qualified Personnel

Technician EG04;Rusts

Biologist, CFIA Plant Diagnostic Lab.

CRTI Post-CRTI

Ray Tropiano

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.