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QBOL:QBOL:

Development of a new diagnostic Development of a new diagnostic

tool using DNA tool using DNA barcodingbarcoding to identify to identify

quarantine organisms in support of quarantine organisms in support of

plant healthplant health

www.qbol.orgwww.qbol.org

peter.bonants@wur.nlpeter.bonants@wur.nl

What is QBOL?What is QBOL?

� > 20 organizations (universities, research institutes and phytosanitary organizations) in 15 countries

� Financed by EU 7th Framework Program� 3 M€ for 3 years

� Developing DNA barcoding to identify quarantine organisms in support of plant health

Why DNA barcoding?Why DNA barcoding?

� Increasing world wide trading of plants enhances risk of spreading harmful organisms

� Decreasing taxonomic knowledge needed to identify Q-organisms

� DNA barcoding offers accurate identificationand focuses on strengthening the link between traditional and molecular taxonomy

Three principle QBOL ObjectivesThree principle QBOL Objectives

� to DNA barcode relevant Q-organisms + morphologically and/or taxonomically

related organisms

� to develop a database of DNA barcode sequences plus relevant

taxonomic/geographic/host data

� to develop a DNA bank for the selected set

of Q-organisms + morphologically and/or taxonomically related organisms

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� Which?

� Fungi

� Arthropods

� Bacteria

� Nematodes

� Viruses

� Phytoplasmas

Targets Targets QuarantineQuarantine

� Council Directive 2000/29/EC

� EPPO list A1 and A2

Phytophthora ramorum

Xanthomonas fragariaePinewood nematode

PolerovirusPhytoplasm

Anoplophora chinensis

Which:

Monilinia, Ceratocystis, Melampsora, Puccinia,Thecaphora and Mycosphaerella

WP2: FungiWP2: Fungi

Monilinia fructicola

Ceratocystis fagacearum, C. fimbriata f. sp. platani, C. virescens

Melampsora farlowii, M. medusae

Mycosphaerella dearnessii (anamorph: Septoria acicola), M. laricis-leptolepidis, M. populorum (=Davidiella populorum; anamorph: Septoria musiva), Septoria lycopersici var. mailagutii (=Septoria malagutii)

In their taxonomic context

WP2: FungiWP2: Fungi

- Internal transcribed spacers of rDNA operon (ITS):

V9G, ITS1, ITS1F / ITS4, LR6

- Beta-tubulin (TUB):

T1, Bt1a / Bt1b, Bt2b

- Cytochrome oxidase I (COI):

PenF1 / PenR1, AspR1

- Histone H3 (HIS):

CYLH3F / CYLH3R

- Translation elongation factor 1-alpha (TEF):

EF1-728F / EF1-986R, EF-2

- Calmodulin (CAL):

CAL-228F / CAL-737R

- Actin (ACT):

ACT-512F / ACT-783R

WP2: Fungi: potential barcode lociWP2: Fungi: potential barcode loci

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� Which?

WP3: ArthropodsWP3: Arthropods

1.1. Number of species (198), multiple target crops Number of species (198), multiple target crops (Agriculture also forests, ornamentals etc.)(Agriculture also forests, ornamentals etc.)

2.2. Example : Example : DiabroticaDiabrotica spp. on Maizespp. on Maize

3.3. Billion $ cost in US, introduced in EuropeBillion $ cost in US, introduced in Europe

4.4. Pesticide use (20 to 25 million acres in US) Pesticide use (20 to 25 million acres in US) ��indirect costs hardly estimatedindirect costs hardly estimated

5.5. Species complex (i.e. Ips, Species complex (i.e. Ips, GonipterusGonipterus, , EpitrixEpitrix, , BemisiaBemisia globaly poorly known)globaly poorly known)

Cox1 ITS

QBOL : Barcoding Quarantine Pests

Id

Collect

Acleris gloverana (Walsingham)Acleris variana (Fernie)Aculops fuchsiae KeiferAeolesthes sarta (Solsky)

Agrilus planipennis FairmaireAleurocanthus spiniferus (Quaintance)Aleurocanthus woglumi AshbyAnastrepha obliqua (Macquart)

Anastrepha fraterculus WiedmannAnastrepha ludens (Loew)Anastrepha suspensa LoewBlitopertha orientalis (Waterhouse)

Anoplophora chinensis (Thomson)Anoplophora glabripennis (Motschulsky)Anthonomus bisignifer SchenklingAnthonomus eugenii Cano

Anthonomus grandis BohemanAnthonomus quadrigibbus SayAnthonomus signatus SayAonidiella citrina (Coquillett)

Arrhenodes minutus (Drury)Aschistonyx eppoi InouyeAulacaspis yasumatsui TakagiBactrocera cucumis (French)

Bactrocera cucurbitae CoquillettBactrocera dorsalis (Hendel)Bactrocera invadens (Hendel)Bactrocera minax (Enderlein)

Bactrocera tryoni (Froggatt)Bactrocera tsuneonis (Miyake)Bactrocera zonata (Saunders)Bemisia tabaci (Gennadius) & biotype B

Cacoecimorpha pronubana HübnerCacyreus marshalli ButlerCarneocephala fulgida NottinghamCarposina niponensis (Walsingham)

Cephalcia lariciphila (Wachtl)Ceratitis capitata WiedemannCeratitis quinaria (Bezzi)Ceratitis rosa Karsch

Ceratitis cosyra (Walker)Choristoneura conflictana (Walker)Choristoneura fumiferana (Clemens)Choristoneura occidentalis Freeman

Choristoneura rosaceana (Harris)Circulifer tenellus (Baker)

Species listWP3 Barcoding Arthropods

OBJECTIVES

WP3 Barcoding ArthropodsField trips and contacts North and South America

1. Nearly half the Q arthropods occur in Americas --> Focus

2. Trips to Costa Rica (Focus on Q pests of Citrus, palm trees and fruit trees)

3. Visit to USDA Fort Pierce (David Hall) ) - Q pests Vitis, Citrus and ornamentals

4. Trip to Guadeloupe (Focus on Fruit flies, whiteflies, Agromyzids)

5. Trip to Canada (Epitrix and other Q pests)

6. Contact and initiating collaboration with Norman Barr (USDA - Aphis Texas). Activate the Quad WG

Field tripsFruitful contactsOther contacts

WP3 Barcoding ArthropodsField trips and contacts North and South America

Epitrix sp

Homalodisca coagulata Aleurocanthus woglumi

Liromyza sativaeRhynchophorus palmarum

Diaphorina citri

See for complete list of targets the QBOL website

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WP4:WP4: BacteriaBacteriaQBOL

Priority group 1

QBOL Priority group 2

Xylella fastidiosa X

Clavibacter michiganensis subsp. sepedonicus X

Pseudomonas solanacearum = Ralstonia solanacearum

X

3 phylotypesXanthomonasstrains pathogenic to Citrus X

X

X

3 subspp.

Xanthomonas oryzaepv. oryzae& oryzicola X

X

2 pvs

Clavibacter michiganensis subsp. insidiosusX

Clavibacter michiganensis subsp. michiganensis X

Xanthomonas axonopodis pv. phaseoli X

Xanthomonas vesicatoria& X. axonopodispv. vesicatoria

X

XXanthomonas fragariae X

Xanthomonas translucens X

Xanthomonas axonopodispv. dieffenbachiae X

WP4WP4: Bacteria: potential barcode loci: Bacteria: potential barcode loci

- 16S

- GyrB : DNA gyrase, subunit β, which unwinds double stranded DNA

- rpoB : RNA polymerase, subunit β, involved in RNA biosynthesis

WP5:WP5: NematodesNematodesD 5.1 List of selected nematode Q-organisms (Month 6)

SG1 SG2 SG3 1 2 3X Meloidogyne arenaria Aphelenchoides besseyi Anguina tritici Longidorus macrosoma

X Meloidogyne incognita Ditylenchus destructor Aphelenchoides ritzemabosi Meloidogyne exigua

X Meloidogyne javanica Ditylenchus dipsaci Aphelenchoides bicaudatus Paratrichodorus porosus

X X Meloidogyne enterolobii/mayaguensis Globodera pallida Aphelenchoides saprophilus Paratrichodorus anemones

X X Meloidogyne chitw oodi Globodera rostochiensis Bursaphelenchus conicaudatus Paratrichodorus pachydermus

X X Meloidogyne fallax Heterodera glycines Bursaphelenchus fraudulentus Pratylenchus loosi

X Meloidogyne hapla – race A Hirschmaniella spp. Ditylenchus adasi Pratylenchus mediterraneus

X Meloidogyne minor Longidorus diadecturus Globodera achilleae Pratylenchus crenatus

X Meloidogyne naasi Meloidogyne ardenensis Globodera tabacum Pratylenchus neglectus

X Bursaphelenchus xylophilus Meloidogyne maritima Heterodera avenae Pratylenchus penetrans

X Bursaphelenchus mucronatus Nacobbus aberrans Heterodera f ilipjevi Pratylenchus thornei

X Bursaphelenchus chengi Radopholus similis (attacking

citrus, formerly R. citrophilus)

Heterodera latipons Pratylenchus vulnus

X Bursaphelenchus doui Heterodera schachtii Radopholus arabocoffeae

X Bursaphelenchus thailandae Radopholus similis (not

attacking citrus)

Helicotylenchus dihystera Radopholus duriophilus

X Xiphinema americanum sensu lato Helicotylenchus pseudorobustus Subanguina radicicola

X Xiphinema diversicaudatum Xiphinema bricolense Helicotylenchus varicaudatus Trichodorus cedarus

X Xiphinema rivesi Xiphinema californicum Helicotylenchus vulgaris Trichodorus cylindricus

Hirschmaniella gracilis Trichodorus primitivus

Laimaphelenchus penardi Xiphinema incognitum

Longidorus arthensis Xiphinema index

Longidorus dunensis Xiphinema simile

Longidorus elongatus

17 15 43

nematodes supplied by:ACW, WU-Nematology, FERA, ILVO

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WP5: NWP5: Nematodesematodes

Seven barcode regions will be screened in Priority Group 1:

- SSU rDNA, D1-D2 region

- LSU rDNA, D2-D3 region

- IGS2

- COI

- COII

- RNA polymerase II

WP6:WP6: VirusesViruses

- No standard region

- Not culturable

- Relative small genomes

- 90% RNA viruses

- Change of WP description

WP6WP6: Viruses: Viruses

Species Genus List

Genome sequence available

Partner responsible

Isolates available within WP

Arracacha virus B, oca strain

Nepovirus IAI No Fera Yes

Potato black ringspot virus

Nepovirus IAI / EPPO A1 No Fera Yes

Potato virus T Trichovirus IAI /EPPO A1 Yes x2 CIP Yes

Chrysanthemum stem necrosis virus

Tospovirus EPPO A1 No PRI Yes

Potato yellow dwarf virus

Rhabdovirus EPPO A1 No

Potato yellowing virus Alphamovirus EPPO A1 No CIP Yes

Tomato infectious chlorosis virus

Crinivirus EPPO A2 No Fera Yes

Iris yellow spot virus Tospovirus EPPO Alert No PRI Yes

Tomato torrado virus Toradovirus N/A1

Yes PRI Yes

Tomato marchitez virus Toradovirus N/A1

Yes PRI Yes

WP6:WP6: Viruses: Method DevelopmentViruses: Method Development

� Using 454 GS-FLX

� Produces 1 million long reads (500bp)

� Method involves sequencing cDNA from infected plant and identifying non plant sequences

� Initial sample PepMV infected plant

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454 Sample multiplexing

• Using random amplification to produce cDNA

• Introduce MiD into amplification primer• (90% of our sequences require removal of amplification primer sequence)

• Worked with test sample PVY

• 150 MiDs x16 lanes = 2400 samples per run

Fera Targets

• Arracacha virus B (25000 reads av 366bp)

• Potato black ringspot virus (50,299 reads av 374.62bp)

• Tomato infectious chlorosis virus (13,816 reads av 321bp)

WP6WP6: Viruses: Viruses

Andean potato latent virus (APLV)

Andean potato mottle virus (APMoV)

Potato yellowing virus (PYV)

Potato virus T (PVT)

CIP TargetsCIP Targets

WP6WP6: Viruses: Viruses

• 5 isolates of CSNV now available on infected plants

• 2 clearly different isolates of IYSV now available

• Several Isolates of ToTV and ToMarV and new toradovirus Tomato chocolate virus (ToChV) available for seq analysis

PRI TargetsPRI Targets

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WP7WP7: Phytoplasmas: Phytoplasmas

� Cell wall-less bacteria

� Inhabits the plant phloem

� Small genomes (~500-1200 kbp)

� Transmitted by insect vectors

� Obligate parasites

� Symptoms include witches broom, phyllody, virescence, dwarfing, yellowing

WP7WP7: Phytoplasmas: Phytoplasmas

Phytoplasma taxonomy

Mainly based on 16S rDNA

~1200 bp

WP7WP7: Phytoplasmas: potential barcode loci: Phytoplasmas: potential barcode loci

- 16S

- Tuf

- SecA

- 16S-23S spacer

- Ribosomal proteins

- SecY

- rpoC

WP7WP7: Phytoplasmas: which targets?: Phytoplasmas: which targets?

See for complete list of targets the QBOL website

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WP8. DNA bank: ObjectivesWP8. DNA bank: Objectives

To develop protocols for the correct storage and whole genome amplification (WGA) of extracted DNA and RNA samples

To develop a DNA bank, containing DNA/RNA and/or WGA amplified material from selected quarantine and regulated plant pathogens and their taxonomically closely related species.

WP8. DNA bankWP8. DNA bank

� Problems:

� Limited DNA quantities

� Solutions:

� Clones

� Whole Genome Amplification (WGA)

� Useful for non-cultivable organisms or for archived material in collections

WP8 DNA Banks: WGAWP8 DNA Banks: WGA

10 ng

1 ng

WGA

1,2,3,4

Nanodrop

Gel

TaqMan

Sequence

AFLP

WP8. DNA bank: WGA ConclusionsWP8. DNA bank: WGA Conclusions

� GenomePlex discounted - fragmentation

� Rapisome discounted - gene loss

� Repli-G and GenomiPhi mostly equivalent

� Repli-G = equipment requirements and more complex, therefore….

� GenomiPhi selected

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WP8. DNA bank: StorageWP8. DNA bank: Storage

WGA amplified material P. ramorum

A) FTA cards (Whatman)

B) Sample Matrix tubes, (Genvault)

C) DNA Lateral Flow Devices (paper Fera)

D) Qiasafe (Qiagen)

E) Lyophilisation with and without Trehalose

F) TE buffer at –20oC and -80

oC (control)

WP8. DNA bank: StorageWP8. DNA bank: Storage

Storage method

T0=0, T1=3 and T2=6 months

WP9. Database: BioLomicsWP9. Database: BioLomics

Database: www.q-bank.eu

WP 10. ValidationWP 10. Validation

Task 10.1: Validation of barcoding protocols

(DNA/RNA extraction, amplification, sequencing)

20 samples (provided by participants) extracted,

amplified and sequenced according to developed protocols

Task 10.2: Validation of diagnostic use of barcoding

20 DNA/RNA samples (provided by PPS) amplified,

sequenced and database searching according to

developed protocols

Endusers: NPPO’s

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WP11. Dissemination: ObjectivesWP11. Dissemination: Objectives

-To disseminate results obtained within the project to stakeholders.

- To train mandated labs in EU and third countries.

- To organise final workshop and symposium (2012).

WP11 Dissemination: WebsiteWP11 Dissemination: Website

� 1 oct 2009: 1517 visitors

� 1 july 2010: 4101 visitors

www.qbol.org

WP11 Dissemination: Flyer, posterWP11 Dissemination: Flyer, poster WP11 Dissemination: EWP11 Dissemination: E--newsletter externalnewsletter external

� First issue december 2009: Fungi

� Second issue may 2010: Nematodes

� Other subjects

� Stakeholders

� Subscribe via

website

www.qbol.org

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DNA barcode identificationDNA barcode identification QBOL: Barcoding for Plant HealthQBOL: Barcoding for Plant Health

� Genes with sequence difference between Q-organisms and closely related organisms

� Easy to amplify with generic primers

� Culture collections

� Taxonomic experience

� Accessible Database

� Development of ID and DET methods

� Validation – EPPO/IPPC

� Implementation – NPPO’s

Partners QBOLPartners QBOL

www.qbol.orgwww.qbol.org

QBOL: Barcoding for Plant HealthQBOL: Barcoding for Plant Health

� GenVault DNA storage

� Qiagen: DNA/RNA extraction

CollaborationCollaboration

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QBOL: Barcoding for Plant HealthQBOL: Barcoding for Plant Health

Future: DNA barcoders

QBOL: AcknowledgementsQBOL: Acknowledgements

Ewald Groenewald, William Quaedvlieg, Els Verstappen

Jean-Yves Rasplus, Beatrice Courtial, Alain Roques

Martine Maes, Liselot Detemmerman, Kim Heylen, Joanna Zaluga, Paul de Vos

Juerg Frey, Martijn Holterman, Sebastian Kiewnick, Hans Helder, Paul Mooyman

Neil Boonham, Ian Adams, Rachel Glover, Jennifer Hodgetts, Rene vd Vlugt

Assunta Bertaccini, Mogens Nicolaisen

Bart vd Vossenberg, Linda Kox

Vincent Robert, Pedro Crous

Ian Barker, Jan Kreuze, Wilmer Cuellar

Advisory Board: Paul Hebert, Francoise Petter, André Levesque, Paul vd Boogert

YOU???

Thank you for your attention!Thank you for your attention!

To be ….or not to be barcodedTo be ….or not to be barcoded

That’s the question!That’s the question!

www.qbol.orgwww.qbol.org

peter.bonants@wur.nlpeter.bonants@wur.nl