using real-time qpcr to monitor for plant pathogens threatening canada… · using real-time qpcr...

Using real-time qPCR to monitor for plant pathogens threatening Canada’s grain industry

Sarah HambletonBiodiversity (Mycology), Eastern Cereal and Oilseed Research Centre, Ottawa

1.

Introduction

2.

Spore trap network and Asian soybean rust

3.

Other rust pathogens

4.

Scaling up and delving deeper

5.

Summary

Outline

Monitoring: To observe, supervise, or keep under review; to measure or test at intervals, especially for the purpose of regulation or control, or to check or regulate the technical quality of something.

Oxford Dictionary

“. . .surveillance is the process of searching, detection is the process of finding, and diagnosis is the process of determining and/or verifying what is found.”

Stack JP, Fletcher J. Global infectious disease surveillance and

detection. Washington, DC: The National Academies Press; 2007. Plant biosecurity

infrastructure for disease surveillance and diagnostics; pp. 95–102.

Technique

Specificity: probability of

detecting TRUE positives

Specificity: probability of

detecting TRUE negatives

Feasibility for routine

analysis, execution and interpretation

Rapidness Cost

Molecular hybridisation + ++++ ++ + +++

FISH ++ ++ +++ + ++

Conventional PCR +++ ++++ +++ +++ +++

Nested PCR in a single tube ++++ ++++ +++ ++ +++

Multiplex PCR +++ ++++ +++ +++ +++++

Multiplex nested PCR ++++ ++++ ++ +++ ++++

Real-time PCR (Taqman) +++++ +++++ ++++ +++++ +++

NASBA (Molecular Beacon) +++++ ++++ ++++ ++++ ++

LAMP ++++ ++++ +++ ++++ ++

Microarrays + +++++ + ++ +

Excerpt from López, M.M. et al. 2009. Are Molecular Tools Solving the Challenges Posed by Detection of Plant Pathogenic Bacteria and Viruses? Curr. Issues Mol. Biol. 11: 13-46.

Development of DNA assay DNA sequencing

Databases

Collections

Primer & Probe design

Confirmation & monitoring

Bioinformatics

Validation

PCR assay

Optimization

Taxo

nom

y

Adapted from a slide prepared by by

C.A. Lévesque

(AAFC)

RTQ-PCR, Q-PCR, qPCR, qrt-PCRreal-time (quantitative) PCR

vsRT-PCR, RRT-PCR, RT-rtPCR

real-time reverse-transcription PCR

Detection and diagnosis at the same time•

amplify one or more specific sequences in a DNA sample •

simultaneously quantify –

determine absolute number of copies or relative amount when normalized to DNA input or additional normalizing genes

•

use of reference standard provides diagnosis

Real-time qPCR

qPCR – Taqman assay

PCR with the addition of a fluorogenic

gene probe which carries a reporter dye at the

5′-end and a quencher at the 3′-end

From: Schwartz, T. et al. 2007. Anal. Bioanal. Chem. 387:513–521

NB: 3 opportunities to incorporate specificity

2. Spore trap network and Asian soybean rust

• cone-shaped lesions formed primarily on leaf undersurface• lesions gray to tan or reddish brown• infective urediniospores released through pore• spores travel long distances on air currents• alternate host in lifecycle not known• documented on many other legume hosts (35 species in 18 genera) but

recent field studies in Florida/Argentina suggest limited/no risk

Phakopsora pachyrhizi (Asia, Australia, Africa, Central/South America, USA)

Phakopsora meibomiae (Central/South America)

Computational Epidemiology & Aerobiology Laboratory, Penn State(http://www.ceal.psu.edu/SBR_expansion.pdf)

http://www.ceal.psu.edu/SBR_expansion.pdf

Fungicide Trials: Attapulgus, Georgia 2005

Photo: Bob Kemerait, University of GA

Burkard

CycloneAir Sampler

LODA Rainfall Collector

Spore-trapping network in Canada 2007-2010

JB Passive Sampler

Sentinel plot scouting from 2005…

14 sites, weekly processing of 22 samples for 18-22 weeks

2007 Spore trap sites

“puccinia

pathway” phenomenon –

overwintering

in extreme southern US

Major events Location

JB Collector (wet and dry

deposition)LODA (Rain)

Burkard (Air)

Week # (n=32) Installed date Collected date

ONT, St. Thomas PMC191 n/a n/a 7 13/Jun/07 20/Jun/07ONT, St. Thomas PMC235 n/a n/a 10 4/Jul/07 11/Jul/07SK, Saskatoon PMC234 n/a n/a 10 3/Jul/07 10/Jul/07

event 1 - July 9-18 ONT, Kemptville PMC245 n/a n/a 11 11/Jul/07 18/Jul/07event 1 - July 9-18 ONT, Morrisburg PMC246 n/a 11 10/Jul/07 17/Jul/07event 1 - July 9-18 ONT, Ottawa PMC238 LD026 BK032 11 11/Jul/07 18/Jul/07event 1 - July 9-18 ONT, Ridgetown PMC242 LD027 BK033 11 10/Jul/07 17/Jul/07event 1 - July 9-18 ONT, St. Thomas PMC243 n/a n/a 11 11/Jul/07 17/Jul/07event 1 - July 9-18 ONT, Toronto PMC247 LD028 11 9/Jul/07 16/Jul/07event 1 - July 9-18 ONT, Woodslee PMC236 n/a n/a 11 9/Jul/07 16/Jul/07event 1 - July 9-18 SK, Saskatoon PMC241 n/a n/a 11 10/Jul/07 17/Jul/07

ONT, Ottawa PMC278 14 1/Aug/07 8/Aug/07ONT, Woodslee PMC287 n/a n/a 14 2/Aug/07 9/Aug/07ONT, Caledonia PMC293 n/a n/a 15 7/Aug/07 14/Aug/07ONT, Ridgetown LD043 15 7/Aug/07 14/Aug/07

event 2 - Aug 13-28 MB, Morden PMC307 n/a n/a 16 14/Aug/07 21/Aug/07event 2 - Aug 13-28 ONT, Caledonia PMC308 n/a n/a 16 14/Aug/07 21/Aug/07event 2 - Aug 13-28 ONT, Forest PMC310 n/a n/a 16 14/Aug/07 21/Aug/07event 2 - Aug 13-28 ONT, Harrow PMC302 LD044 16 13/Aug/07 20/Aug/07event 2 - Aug 13-28 ONT, Morrisburg LD049 n/a 16 14/Aug/07 22/Aug/07event 2 - Aug 13-28 ONT, Ottawa PMC304, 305, 306 LD045 BK046 16 15/Aug/07 22/Aug/07event 2 - Aug 13-28 ONT, Ridgetown BK047 16 7/Aug/07 21/Aug/07event 2 - Aug 13-28 ONT, Toronto PMC312 n/a 16 14/Aug/07 22/Aug/07event 2 - Aug 13-28 SK, Saskatoon PMC311 n/a n/a 16 14/Aug/07 21/Aug/07event 2 - Aug 13-28 ONT, St. Thomas PMC325 n/a n/a 17 22/Aug/07 29/Aug/07

ONT, Morrisburg PMC339 n/a n/a 18 29/Aug/07 5/Sep/07ONT, Toronto PMC337 n/a 18 29/Aug/07 7/Sep/07ONT, Morrisburg LD085 n/a 26 22/Oct/07 30/Oct/07ONT, Ottawa LD084 26 24/Oct/07 1/Nov/07SK, Saskatoon PMC398 n/a n/a 26 23/Oct/07 30/Oct/07

2007 positive detections of Asian soybean rust

Combined with US

results and compared to predictions

from the Penn State

IAMS

(Integrated Aerobiology

Modeling System)

forecasting model

a. 8 -

14 August 2007 b. 15 -

21 August 2007

c. 22 -

28 August 2007

Isard, S.A. et al. 2010. Predicting seasonal soybean rust incursions into the North American continental interior using sentinel plot monitoring, spore trapping, and aerobiological modeling. Submitted to Phytopathology.

http://sbr.ipmpipe.org/cgi-bin/sbr/public.cgi http://www.ceal.psu.edu/sbrcomparisons.htm

http://sbr.ipmpipe.org/cgi-bin/sbr/public.cgi

http://www.ceal.psu.edu/sbrcomparisons.htm

2. Summary

2007 message: infection can occur in Canada; widespread deposition in mid-July and mid-

to late August after a series of storm fronts moved through. Late season increased field monitoring located infected plants in SW Ontario.

2008 message: long-range transport of spores is possible early in the growing season (deposition across a broad sampling area in late June/early July)

2009/2010 messages: low-risk years, due to environmental factors

Every qPCR

positive reaction was PCR-cloned to verify the diagnosis, contributing to a population genetics study

Simple case:Diagnostics at the species levelInvasive alien, no close relatives in CanadaOther documented legume hosts seemingly not very susceptible

3. Other rust pathogens

Specificity –

detecting true positives and true negatives → assay design needs to be based on adequate sampling→ on-going validation

NB: environmental sample testing of new real-time assay for stripe rust lead to improvement of the assay

Puccinia striiformis(stripe rust of wheat)

PUR N1249 Hordeum

jubatum

USA Aug1993PUR 66275 Triticum

aestivum

USA Jun1981HMAS 79075 Triticum

aestivum

China Aug1996DAOM 240066 Triticum

aestivum

China 2006DAOM 240067 Triticum

aestivum

China Jun2006DAOM 240069 Triticum

aestivum


aestivum

China 2006RS477 Triticum

aestivum


aestivum


aestivum


aestivum

China 2006K 78118 Hordeum

secalinum

UK Jun2000PUR F15603 Elymus

repens

India Mar1950PUR F17111 unknown Turkey Jun1960PUR F19515 Hordeum

sp Argentina Feb1970PUR 60028 Elymus

elymoides

USA Sep1963PUR 61492 Triticum

aestivum

USA Aug1963BPI 0193871 Triticum

aestivum

Afghantan

July1970BPI 0195217 Hordeum

comosum

Argentina Feb1970DAOM 240065 Achnatherum

inebrians

China 1998PUR N5378 Lolium

parenne

USA Aug1992BPI 0199096 Dactylis

glomerata

China Aug1986PUR N5374 Dactylis

glomerata

USA Aug1992K 108141 Dactylis

glomerata

UK Sep1977PUR N1254 Dactylis

glomerata

Canada Aug1994PUR N5383 Dactylis

glomerata

UK Nov1966DAOM 220657 Poa

pratensis

Canada Sep1996PUR N5368 Poa

nemoralis

USA May1956PUR N5354 Poa

secunda

USA Apr1956PUR 59733 Poa

pratensis

Canada Jul1969DAOM 212041 P poa

nemoralisDAOM 189681 P poa

nemoralisPDD 60181 P poa

nemoralisDAOM 240189 P poa

nemoralisDAOM 193284 P poarumDAOM 240188 P poarumBPI 1100377 P poarum

Puccinia striiformis

Puccinia gansensis

Puccinia pseudostriiformis

Puccinia striiformoides

91/97/97

86/96/100

58/<50/<50

95/95/9094/98/100

84/99/10084/91/100

70/92/94

100/100/100

Liu, M. and Hambleton, S. 2010. Fungal Biology 114: 881-899.(CRTI-04 -

0045RD )

Pque-CoxI-lna

Pque-CoxI

Pque-ITS2

Pque-ITS1

Fgra-MAT

Pgra-CoxI

Pgra3-RPB2

Pgra1-RPB2

Pram-Elic

Send-IGS

lmbd800-

L524G

Paln-CoxII-

lna

Paln-CoxII

Paln-ITS2

Paln-ITS1

Ppac-ITS1

Pgra-RPB2

Pgra2-Btub

Pgra1-Btub

Pram-Btub

Send-18S

lmbd800-

U354F

Pinf-Nadh1

-lna

Pinf-Nadh1

Pinf-ITS2

Pinf-ITS1

Cpol-HMG

Pgra-Btub

Pgra4-ITS1

Pgra3-ITS1

Pram-ITS

Send-ITS

lmbd600-

L400E

Pstr4-CoxI

Pstr3-CoxI

Pstr2-CoxI

Gabi5-Btub-lna

Tind-Mit

Pgra2-ITS1

Pgra1-ITS1

Pgra-ITS1

Pcor5-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-3C

Pstr1-Btub

Pstr-Btub

Pstr3-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-Btub

Pcor2-ITS1

Pcor1-ITS1ST1A

1110987654321

Pque-CoxI-lna

Pque-CoxI

Pque-ITS2

Pque-ITS1

Fgra-MAT

Pgra-CoxI

Pgra3-RPB2

Pgra1-RPB2

Pram-Elic

Send-IGS

lmbd800-

L524G

Paln-CoxII-

lna

Paln-CoxII

Paln-ITS2

Paln-ITS1

Ppac-ITS1

Pgra-RPB2

Pgra2-Btub

Pgra1-Btub

Pram-Btub

Send-18S

lmbd800-

U354F

Pinf-Nadh1

-lna

Pinf-Nadh1

Pinf-ITS2

Pinf-ITS1

Cpol-HMG

Pgra-Btub

Pgra4-ITS1

Pgra3-ITS1

Pram-ITS

Send-ITS

lmbd600-

L400E

Pstr4-CoxI

Pstr3-CoxI

Pstr2-CoxI

Gabi5-Btub-lna

Tind-Mit

Pgra2-ITS1

Pgra1-ITS1

Pgra-ITS1

Pcor5-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-3C

Pstr1-Btub

Pstr-Btub

Pstr3-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-Btub

Pcor2-ITS1

Pcor1-ITS1ST1A

1110987654321

each box = one probe for one geneOrange

= P. striiformis (11 probes)Pink

= P. coronata (14 probes)Green = P. graminis (12 probes)Teal Blue = reaction controls (A1/A11)Red-Brown

= universal standard (B1/C1)

DNA array hybridization with membrane-bound markers was tested for multiple species detection in a single assay.

Preliminary results for first round testing with P. striiformis DNA

Both reaction controls and I universal standard were positive; 9 of 11 Ps probes were positive but 2 failed (pink box –

includes A11 reaction control).Two strong cross reactions need investigation and optimization (yellow circles).

4. Scaling up: Macroarray for multiple taxa

4. Delving deeper – biodiversity baseline

We have a bank of DNA extracts, collected over 4 years…..and counting…

This resource can be used for validation of new assays and mined

for historical

presence /absence information for air-borne pathogens.

We are now expanding our network to include sites in BC and PEI,

and are expanding our scope of interest.

For selected sites we will apply next-generation 454 pyrosequencing

technology to ribosomal DNA (rDNA) amplicons

derived from archived and new air and rain samples.

This will allow us to develop a baseline profile of harmful and beneficial fungi and bacteria from 2007 to 2012

Pilot run using a qPCR-positive 2008 air sample for soybean rust47,500 sequences –

bioinformatics pipeline is in developmentP. pachyrhizi -

like sequences were recovered

5. Summary

For species level diagnostics –

qPCR

Taqman

assays are becoming very common.

Accuracy and reproducibility depends on accurate taxonomic and ecological information in the development stage.

For many pathogens though, genome data will be necessary to find

markers diagnostic at the appropriate level –

pathogenicity

/ race level will be hardest of all.

Baseline biodiversity data will help us to recognize new incursions or variants.

Cereals: BarleyOatsRyeTriticaleWheat

Selected published qPCR assays

Pulses: beanschick peasfaba

beans lentilspeas

Other crops: buckwheat corn


Oilseeds:canola flaxseedmustardrapeseedsafflower seedsolinsoybeanssunflower seed


Spore trap Network Collaborators / Funding:US Department of Agricuture (USDA)Univeristy of MinnesotaReid FrederickLes SzaboCharlie BarnesJerry Johnson

Canadian Food Inspection Agency (CFIA)Stéphen Brière

Grain Farmers of Ontario (GFO) / Ontario Soybean Rust CoalitionCrosby Devitt, Alison Walden-Coleman

Manitoba Pulse Growers (funds to R. Conner)

AAFC Pest Management Centre(Pesticide Risk Reduction Strategies)Cezarina Kora

Le ministère de l'Agriculture, des Pêcheries et de l'Alimentation du Québec (MAPAQ)Claude Parent

Agriculture and Agri-Food Canada (AAFC)Ottawa Kathryn Oliver

Hanaa SaeedRaymond TropianoFrançois Meloche

Harrow Terry AndersonMorden Robert ConnerSaskatoon Bruce GossenAlberta Kelly Turkington

ON Ministry of Agriculture, Food & Rural Affairs (OMAFRA)Ridgetown: Albert Tenuta

Cheryl Van HerkKemptville: Gilles Quesnel

University of TorontoBruce Hall

MAPAQNadia SurdekÈve Cayer

using real-time qpcr to monitor for plant pathogens threatening canada… · using real-time qpcr...

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