QBOL, DNA barcodes to identify phytobacteria subjected to EU quarantine regulations

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QBOL, DNA barcodes to identify

phytobacteria subjected to EU

quarantine regulations

Cottyn B., L. Detemmerman, M. Maes

COST873 - Annual Meeting

13 - 15 September 2010

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Introduction

AIM: develop new diagnostic tool for plant health



identification of quarantine organisms using DNA barcoding

DNA 'barcode„ = short genomic DNA sequence for species identification

Financed by 7th Framework Program of

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Introduction

Barcoding of Fungi

Barcoding of Arthropods

Barcoding of Bacteria

Barcoding of Nematodes

Barcoding of Viruses

Barcoding of Phytoplasmas

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QBOL bacteria

Partners:

• Institute for Agricultural and Fisheries Research (ILVO, Belgium)

• Laboratory of Microbiology, Ghent University (LM-UGent, Belgium)

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QBOL bacteria

Activities

1. Design

DNA barcode

for quarantine bacteria

2. Develop

standard protocol

for DNA isolation, amplification and sequencing

3. Set up

public barcode-database

, linked to biological information

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Target Q-organism Priority 1 Priority 2 Partner

Xylella fastidiosa X ACW

Clavibacter michiganensis subsp. sepedonicus X LM-UGent

Ralstonia solanacearum 3 phylotypes ACW

Xanthomonads pathogenic to Citrus 3 subsp. ILVO

Xanthomonas oryzae 2 pv's ILVO

Clavibacter michiganensis subsp. insidiosus X LM-UGent

Clavibacter michiganensis subsp. michiganensis X LM-UGent

Xanthomonas axonopodis pv. phaseoli X ILVO

Xanthomonas vesicatoria X ILVO

Xanthomonas axonopodis pv. vesicatoria X ILVO

Xanthomonas fragariae X ILVO

Xanthomonas translucens X ILVO

Xanthomonas axonopodis pv. dieffenbachiae X ILVO

Xanthomonas axonopodis pv. allii X ILVO

Target pathogens

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QBOL @ ILVO

KEY WORK:

• Strain-collection: target xanthomonads & close relatives

• DNA barcode design: discrimination between target xanthomonads & relatives

EXPECTED: Multi-locus DNA barcode for accurate pathogen identification

Gyrase B

gene for phylogeny of

Xanthomonas

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SEQUENCING-STRATEGY

1. 16S rRNA

gene



“genus level”:

Xanthomonas

affiliation

2. Gyrase B



“species level”: phylogenetic identity

3. Additional genes



“subspecies/pathovar level” (work in progress)

gyrB DNA gyrase subunit B

dnaK heat shock protein 70, molecular chaperone

lepA GTP-binding protein

lrp leucine responsive regulatory protein

fyuA tonB-dependent receptor, structural gene

hrpB type III secretion system

hrpE type III secretion system

hrpF type III secretion system

AvrBs2 avirulence protein

pthA avirulence protein, AvrBs3 family

XopN type III effector protein

Core genes

Accessory genes

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gyrB

phylogeny

Parkinson

et al.

, 2007

(530 bp sequences)

(pv‟sdieffenbachiae, phaseoli ) X. melonis

X. citri pv‟s dieffenbachiae, citri, aurantifolii, phaseoli

X. vasicola

X. oryzae

species level clade 1

X. fragariae

X. pisi

X. vesicatoria X. arboricola X. hortorum X. campestris X. cucurbitae X. codiaei

species level clade 3 species level clade 4

non pathogenic xanthomonasstrains species level clade 7

X. sacchari

X. translucens

X. translucens 99 90 99 95 99 99 99 99 28 63 99 99 99 99 99 99 99 99 98 97 99 93 99 98 78 86 73 63 99 88 99 35 41 89 88 86 72 99 97 67 78 99 0.02 X. euvesicatoria sp. complex

X. euvesicatoria sister clade

X. citri

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xanthomonads on

Citrus

! Classificatie is dynamisch en bepaalt de naamgeving !

Xanthomonas campestris

pv.

citri

“Xanthomonas axonopodis

pv.

citri”

canker

bacterial spot

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xanthomonads on

Citrus

! Classificatie is dynamisch en bepaalt de naamgeving !

Xanthomonas campestris

pv.

citri

“Xanthomonas axonopodis

pv.

citri”

canker

bacterial spot

X. citri

subsp.

citri

X. fuscans

subsp.

aurantifolii

X. alfalfae

subsp.

citrumelonis

(Schaad

et al.

, 2006)

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xanthomonads on

Citrus

! Classificatie is dynamisch en bepaalt de naamgeving !

Xanthomonas campestris

pv.

citri

“Xanthomonas axonopodis

pv.

citri”

canker

bacterial spot

X. citri

pv.

citri

X. citri

pv.

aurantifolii

X. alfalfae

subsp.

citrumelonis

(Ah-You

et al.

, 2009)

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X. alfalfae

subsp.

citrumelonis

X. fragariae X. vesicatoria X. translucens X. melonis X. vasicola

X. pisi

X. arboricola X. hortorum

X. campestris X. cucurbitae

X. codiaei

X. sacchari

X. citri

X. oryzae

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X. citri

pv.

citri

X. citri

pv.

aurantifolii

X. pisi

X. codiaei

X. sacchari

99 90 99 95 99 99 28 99 99 99 99 99 99 99 99 98 97 99 93 99 98 78 86 73 63 99 88 99 35 41 89 88 86 72 99 97 67 78 99 X. euvesicatoria sp. complex

X. citri

X. oryzae

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X. fragariae X. vesicatoria

X. translucens X. melonis

X. vasicola

X. pisi

X. codiaei

X. sacchari

X. citri

X. oryzae

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X. vasicola

X. pisi

X. codiaei

X. sacchari

X. citri

X. oryzae

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X. vasicola

X. pisi

X. codiaei

X. sacchari

X. citri

X. oryzae

(pv‟sdieffenbachiae, phaseoli)

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X. vasicola

X. pisi

X. codiaei

X. sacchari

X. citri

X. oryzae

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X. vasicola

X. pisi

X. codiaei

X. sacchari

X. citri

X. oryzae

“pv. dieffenbachiae”

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gyrB

phylogeny

X. pisi

X. codiaei

X. sacchari

X. citri

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Pathovar resolution

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Conclusion

gyrB

-barcode:

• OK at “species level”: X

. oryzae

,

X. fragariae

,

X. vesicatoria

,

X . translucens

• Insufficient at “subspecies/pathovar level”

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Conclusion

Supplementary loci?

• Core genes:

+ phylogenetic relevance

-

resolution?

• Accessory genes:

+ direct link to

“pathovar”

- horizontal gene transfer

- limited distribution

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Conclusion

Pathovars = “special purpose classification”

(Provisional solution for communication, regulation and quarantine)

• Not covered by „International Code of Names of Prokaryotes‟ (Lapage

et al.

, 1975)

„Approved Lists‟ (Skerman

et al.

, 1980)

• Taxonomically not well investigated

• Sometimes composed of multiple unrelated genotypes (biological entities ?)

• Xanthomonas

classification very dynamic

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