using real-time qpcr to monitor for plant pathogens threatening canada… · using real-time qpcr...
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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)
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
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
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
China 2006DAOM 240070 Triticum
aestivum
China 2006RS477 Triticum
aestivum
China 2006DAOM 240071 Triticum
aestivum
China 2006RS479 Triticum
aestivum
China 2006RS480 Triticum
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.
Pulses: beanschick peasfaba
beans lentilspeas
Other crops: buckwheat corn
Selected published qPCR assays
Oilseeds:canola flaxseedmustardrapeseedsafflower seedsolinsoybeanssunflower seed
Selected published qPCR assays
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