QBOL:

Development of a new diagnostic tool

using DNA barcoding to identify
quarantine organisms in support of
plant health

www.qbol.org
peter.bonants@wur.nl
Introduction
 Plant Research International
 Wageningen University & Research Centre
 Netherlands
 Identification & detection: molecular methods
 Quarantine plant pathogenic species
What is QBOL?

 > 20 organizations (universities, research

institutes and phytosanitary organizations) in 15
countries
 Financed by EU 7th Framework Program
 3 M€ EU + 1 M€ own contribution for 3 years
 Developing DNA barcoding to identify quarantine
organisms in support of plant health
Why DNA barcoding?

 Increasing world wide trading of plants

enhances risk of spreading harmful organisms
 Decreasing taxonomic knowledge to identify Q-
organisms
 Result in significant possible economic damage
 DNA barcoding offers accurate identification
and focuses on strengthening the link between
traditional and molecular taxonomy
Three 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
 Phytophthora ramorum

Targets Quarantine
 Which?
 Fungi
 Arthropods
Anoplophora chinensis
 Bacteria
 Nematodes
Xanthomonas fragariae
 Viruses
Pinewood nematode
 Phytoplasmas

Phytoplasm Polerovirus

 Council Directive 2000/29/EC

 EPPO list A1 and A2
Partners QBOL www.qbol.org
 WP2 leader: Ewald Groenewald (KNAW-CBS)
WP2: Fungi
Which:
Monilinia, Ceratocystis, Melampsora, Puccinia,
Thecaphora and Mycosphaerella
List Q- species and relatives on website
WP2: Fungi: potential barcode loci
- 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
 WP3 leader: Jean-Yves Rasplus (INRA)
WP3: Arthropods
 Which?
1. Number of species (198), multiple target crops
(Agriculture also forests, ornamentals etc.)
2. Example : Diabrotica spp. on Maize
3. Billion $ cost in US, introduced in Europe
4. Pesticide use (20 to 25 million acres in US) 
indirect costs hardly estimated
5. Species complex (i.e. Ips, Gonipterus, Epitrix,
Bemisia globaly poorly known)
 Species list
Acleris gloverana (Walsingham) WP3 Barcoding
Acleris variana (Fernie)
Aculops fuchsiae Keifer
Aeolesthes sarta (Solsky)
Arthropods
Agrilus planipennis Fairmaire
Aleurocanthus spiniferus (Quaintance)
Aleurocanthus woglumi Ashby
OBJECTIVES
Anastrepha obliqua (Macquart)
Anastrepha fraterculus Wiedmann
Anastrepha ludens (Loew)
Anastrepha suspensa Loew
Blitopertha orientalis (Waterhouse) Id Cox1 ITS
Anoplophora chinensis (Thomson)
Collect
Anoplophora glabripennis (Motschulsky)
Anthonomus bisignifer Schenkling
Anthonomus eugenii Cano
Anthonomus grandis Boheman
Anthonomus quadrigibbus Say
Anthonomus signatus Say
Aonidiella citrina (Coquillett)
Arrhenodes minutus (Drury)
Aschistonyx eppoi Inouye
Aulacaspis yasumatsui Takagi
Bactrocera cucumis (French)
Bactrocera cucurbitae Coquillett
Bactrocera dorsalis (Hendel)
Bactrocera invadens (Hendel)
Bactrocera minax (Enderlein)
Bactrocera tryoni (Froggatt)
Bactrocera tsuneonis (Miyake)
Bactrocera zonata (Saunders)
Bemisia tabaci (Gennadius) & biotype B
Cacoecimorpha pronubana Hübner
Cacyreus marshalli Butler
Carneocephala fulgida Nottingham
Carposina niponensis (Walsingham)
Cephalcia lariciphila (Wachtl)
Ceratitis capitata Wiedemann
Ceratitis quinaria (Bezzi)
Ceratitis rosa Karsch
Ceratitis cosyra (Walker)
Choristoneura conflictana (Walker)
Choristoneura fumiferana (Clemens)
Choristoneura occidentalis Freeman
Choristoneura rosaceana (Harris) QBOL : Barcoding Quarantine Pests
Circulifer tenellus (Baker)
WP4: Bacteria
QBOL QBOL
Priority Priority
group 1 group 2
Xylella fastidiosa X
Clavibacter michiganensis subsp.
X
sepedonicus
Pseudomonas solanacearum = Ralstonia X
solanacearum 3
phylotypes
Xanthomonas strains pathogenic to X
Citrus X
X
3 subspp.
Xanthomonas oryzae pv. oryzae & X
oryzicola X
2 pvs
Clavibacter michiganensis subsp.
X
insidiosus
Clavibacter michiganensis subsp.
X
michiganensis
Xanthomonas axonopodis pv. phaseoli X
Xanthomonas vesicatoria & X. X
axonopodis pv. vesicatoria X
Xanthomonas fragariae X
Xanthomonas translucens X
Xanthomonas axonopodis pv.
X
dieffenbachiae
Xanthomonas axonopodis pv. allii
 WP4 leader: Martine Maes (ILVO)

WP4: 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 leader: Juerg Frey (ACW)
WP5: Nematodes
 D 5.1 List of selected nematode Q-
organisms (Month 6)
SG1 SG2 SG3 1 2 3
X 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 chitwoodi 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 filipjevi Pratylenchus thornei

X Bursaphelenchus chengi Radopholus similis (attacking Heterodera latipons Pratylenchus vulnus

X Bursaphelenchus doui citrus, formerly R. citrophilus) Heterodera schachtii Radopholus arabocoffeae

X Bursaphelenchus thailandae Radopholus similis (not Helicotylenchus dihystera Radopholus duriophilus

X Xiphinema americanum sensu lato attacking citrus) 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

nematodes supplied by: Laimaphelenchus penardi Xiphinema incognitum

ACW, WU-Nematology, FERA, ILVO Longidorus arthensis

Longidorus dunensis
Xiphinema index

Xiphinema simile

Longidorus elongatus

17 15 43
WP5: Nematodes
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 leader: Neil Boonham (Fera)
WP6: Viruses

- No standard region
- Not culturable
- Relative small genomes
- 90% RNA viruses
- Whole genome sequence
WP6: Viruses
Genome Isolates
Partner
Species Genus List sequence available
responsible
available within WP
Arracacha virus B, oca
Nepovirus IAI No Fera Yes
strain

Potato black ringspot

Nepovirus IAI / EPPO A1 No Fera Yes
virus

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

Chrysanthemum stem
Tospovirus EPPO A1 No PRI Yes
necrosis virus

Potato yellow dwarf

Rhabdovirus EPPO A1 No
virus

Potato yellowing virus Alphamovirus EPPO A1 No CIP Yes

Tomato infectious
Crinivirus EPPO A2 No Fera Yes
chlorosis virus

Iris yellow spot virus Tospovirus EPPO Alert No PRI Yes

1
Tomato torrado virus Toradovirus N/A Yes PRI Yes
1
Tomato marchitez virus Toradovirus N/A Yes PRI Yes
 WP7 leader: Mogens Nicolaisen (UA)
WP7: Phytoplasmas
Quarantine Phytoplasma species
– Strawberry witches’ broom (16SrI-C)
– ’Witches broom’ on Citrus and few others ’Ca. P. aurantifolia’
(WBDL, 16SrII-B)
– Peach rosette (16SrIII)
– Peach X (16SrIII-A, strains CX and WX)
– Peach yellows (16SrIII)
– Palm lethal yellowing (16SrIV)
– Grapevine flavescence doreé (16SrV-C and 16SrV-D)
– Elm phloem necrosis (16SrV-A, strain EY)
– Apple proliferation ’Ca. P. mali’ (16SrX-A strains AP, AT)
– Apricot chlorotic leafroll ’Ca. P. prunorum’ (16SrX-B, ESFY)
– Pear decline ’Ca. P. pyri’ (16SrX-C, PD)
– Potato stolbur (16SrXII-A)
– Potato purple top wilt (aster yellows) (EPPO list)
WP7: Phytoplasmas: potential barcode loci

- 16S
- Tuf
- SecA
- 16S-23S spacer
- Ribosomal proteins
- SecY
- rpoC
WP2-7: ‘DNA barcodes’

QBOL sequences obtained 3-12-2010

# sequences Remark
WP2 FUNGI 3923 7 loci
WP3 ARTHROPODS 1221 2 loci
WP4 BACTERIA 1646 20 loci
WP5 NEMATODES 720 6 loci
WP6 VIRUSES 11 whole genome seq
WP7 PHYTOPLASMS 126 2 loci

TOTAL 7647
Decision
scheme
 WP8 leader: Peter Bonants (PRI)
WP8: ‘DNA bank’
Development protocols for WGA
whole genome amplification
Development protocols for storage and transport of DNA
 WP9 leader: Vincent Robert (KNAW-CBS)
WP9. Database: BioLomics
 WP10 leader: Bart van den Vossenberg (PPS)
WP10. Validation / Evaluation
- Validation samples
- Ringtest
DNA extraction
Generic PCR amplification
Sequence analysis
Database searching www.q-bank.eu
- Partners from all over the world
- Evaluation DNA barcode as Diagnostic Tool
 QBOL: 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

www.qbol.org
DNA barcode identification

Q-bank database
 Project 2
QBOL

Q B A N K

Project 3
FES Project 1
2006 2008 2010 2012 2014 2016

www.q-bank.eu
 What is Q-bank?
- Database on regulated plant pathogens
General information: distribution, biological, ecological, literature references;
Collection data: geographical data, collection type, status, specimens and/or
isolate;
Nomenclatural data: taxonomy, systematic position;
Morphological data: photographs, characteristics of various taxonomical levels;
Diagnostic data: description of symptoms, illustrations, protocols;
Sequence data: sequences of DNA barcode genes relevant to a specific group.

www.q-bank.eu
 Current state
Fungi: Phoma (281 species) Colletotrichum (21 species, 98 strains) and the Oomycete
genus Phytophthora (c. 100 species, 350 strains/isolates).
Bacteria: (3056 strains): Pseudomonads, Xanthomonads, Erwiniae, Agrobacteria,
Coryneforms and others;
Invasive Plants: (160 species) with special attention to aquatic (non-marine) plants
Nematodes: (120 species; 1273 items): Meloidogyne spp. (root-knot nematodes),
Heterodera and Globodera;
Insects: (233 species; 2754 specimens): Diptera (Tephritidae), Lepidoptera
(Spodoptera), Hemiptera - Aleyrodidae (Bemisia) and Coleoptera - Cerambycidae
(Anoplophora, Monochamus);
Viruses: (90 species; 134 items): belonging to the genera Nepovirus, Potexvirus,
Potyvirus, and Tospovirus, and viroids belonging to the genus Pospiviroid.
www.q-bank.eu
 Ambition
• Q-bank is a world-wide acknowledged database for identity and
reference of plant pathogenic Q-organisms

• Q-bank is an online freely accessible database

• Q-bank is connected to vouchered specimens in public

available culture collections

• Q-bank will be connected to EPPO and CABI databases

www.q-bank.eu
 Unique Characteristics
• Independent and science based curated database
• Link to publicly accessible and curated culture, specimen and DNA
reference collections world-wide, thus making the information
verifiable and replicable.

• Instrumental to polyphasic (pathology/morphology/molecular)

identification; all including intraspecific variation and look-alike's
etc

www.q-bank.eu
 Future
• Q-bank is used by: NPPO’s, mandated and routine diagnostic
laboratories, R&D organisations.

• Q-bank is also used by: public/private organisations in the

production and trade chain.

• Q-bank is a database for all information regarding the identity and

reference of regulated and non-regulated plant pests and
links reference collections.

• Q-bank is self-supporting by acquiring the necessary funds for

maintenance and development.
www.q-bank.eu
 Management Structure
Data-Curatorium phytopathology
Insects

Data-Curatorium
Viruses

Data-Curatorium
Fungi

Steering Data-Curatorium
Committee Coordinator Bacteria
taxonomy collections
Data-Curatorium
Nematodes
Administrator
Data-Curatorium
Phytoplasma's

www.q-bank.eu Data-Curatorium
Invasive plants
www.q-bank.eu
www.q-bank.eu
www.q-bank.eu
www.q-bank.eu
www.q-bank.eu
www.q-bank.eu
 Do you want to collaborate?
Please contact us at : www.q-bank.eu

www.q-bank.eu
 Acknowledgements:
QBOL / Q-bank partners:
Ewald Groenewald, William Quaedvlieg, Els Verstappen, Annette Dullemans,
Jean-Yves Rasplus, Marie-Anne Auger-Rozenberg, 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, Janes Woodhall,
Rene vd Vlugt, Assunta Bertaccini, Mogens Nicolaisen, Olga Makarova, Bart vd
Vossenberg, Linda Kox, Mariette Edema, Antoon Loomans, Annelien Roenhorst,
Evelyn van Heese, Maria Bergsma, Vincent Robert, Pedro Crous, Jan Kreuze,
Giovanna Muller, Wilmer Cuellar,
Validation Partners: NIB, SPA, LNPV, CAIQ, LU, CNPMF, DAFS, INIA, US
Advisory Board: Paul Hebert, Francoise Petter, André Levesque, Paul vd Boogert
To be barcoded or not to barcoded?
..... that is the question!

Thank you for your attention!

www.qbol.org
www.q-bank.eu
peter.bonants@wur.nl