QBOL, DNA barcodes to identify phytobacteria subjected to EU quarantine regulations
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1 QBOL, DNA barcodes to identify phytobacteria subjected to EU quarantine regulations Cottyn B., L. Detemmerman, M. Maes COST873 - Annual Meeting September 2010
2 Introduction Financed by 7th Framework Program of European Union AIM: develop new diagnostic tool for plant health identification of quarantine organisms using DNA barcoding DNA 'barcode = short genomic DNA sequence for species identification
3 Introduction Barcoding of Fungi Barcoding of Arthropods Barcoding of Bacteria Barcoding of Nematodes Barcoding of Viruses Barcoding of Phytoplasmas
4 QBOL bacteria Partners: Institute for Agricultural and Fisheries Research (ILVO, Belgium) Laboratory of Microbiology, Ghent University (LM-UGent, Belgium) Agroscope Changins-Waedenswil Research Station (ACW, Switzerland)
5 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 4. Create DNA-bank of barcoded quarantine bacteria and relatives
6 QBOL bacteria Target pathogens 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
7 ILVO KEY WORK: Strain-collection: target xanthomonads & close relatives DNA barcode design: discrimination between target xanthomonads & relatives Gyrase B gene for phylogeny of Xanthomonas = first barcoding region EXPECTED: Multi-locus DNA barcode for accurate pathogen identification
8 ILVO 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) Core genes 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 Accessory genes 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
9 Parkinson et al., 2007 (530 bp sequences) gyrb phylogeny X. euvesicatoria sp. complex (X. alfalfae subsp. citrumelonis, pv s dieffenbachiae, vesicatoria, allii, ) 67 X. euvesicatoria sister clade (pv s dieffenbachiae, phaseoli ) X. melonis 72 X. citri X. citri pv s dieffenbachiae, citri, aurantifolii, phaseoli X. vasicola X. oryzae X. pisi X. arboricola X. hortorum species level clade 1 species level clade 2 X. campestris X. cucurbitae X. codiaei species level clade 3 species level clade 4 X. fragariae X. vesicatoria 90 non pathogenic xanthomonas strains species level clade 7 X. sacchari species level clade species level clade 6
10 xanthomonads on Citrus! Classificatie Xanthomonas is dynamisch campestris en bepaalt pv. de citri naamgeving! Xanthomonas axonopodis pv. citri canker bacterial spot pathotype A pathotype B, C pathotype E
11 xanthomonads on Citrus! Classificatie Xanthomonas is dynamisch campestris en bepaalt pv. de citri naamgeving! Xanthomonas axonopodis pv. citri canker bacterial spot X. citri subsp. citri X. fuscans subsp. aurantifolii (Schaad et al., 2006) X. alfalfae subsp. citrumelonis
12 xanthomonads on Citrus! Classificatie Xanthomonas is dynamisch campestris en bepaalt pv. de citri naamgeving! Xanthomonas axonopodis pv. citri canker bacterial spot X. citri pv. citri X. citri pv. aurantifolii (Ah-You et al., 2009) X. alfalfae subsp. citrumelonis
13 gyrb phylogeny X. euvesicatoria sp. complex X. alfalfae subsp. citrumelonis 67 X. euvesicatoria sister clade X. melonis 72 X. citri X. vasicola X. oryzae X. pisi X. arboricola X. hortorum species level clade 1 X. fragariae species level clade 2 X. vesicatoria X. campestris X. cucurbitae X. codiaei species level clade 3 species level clade 4 90 non pathogenic xanthomonas strains 95 species level clade 7 X. sacchari species level clade 5 species level clade 6
14 gyrb phylogeny X. euvesicatoria sp. complex 67 X. euvesicatoria sister clade 72 X. melonis X. citri X. citri pv. citri X. citri pv. aurantifolii X. vasicola X. oryzae X. pisi X. arboricola X. hortorum species level clade 1 X. fragariae species level clade 2 X. vesicatoria X. campestris X. cucurbitae X. codiaei species level clade 3 species level clade 4 90 non pathogenic xanthomonas strains 95 species level clade 7 X. sacchari species level clade 5 species level clade 6
15 gyrb phylogeny X. euvesicatoria sp. complex 67 X. euvesicatoria sister clade X. melonis 72 X. citri X. vasicola X. oryzae X. pisi X. arboricola X. hortorum species level clade 1 X. fragariae species level clade 2 X. vesicatoria X. campestris X. cucurbitae X. codiaei species level clade 3 species level clade 4 90 non pathogenic xanthomonas strains 95 species level clade 7 X. sacchari species level clade 5 species level clade 6
16 gyrb phylogeny X. euvesicatoria sp. complex 67 X. euvesicatoria sister clade X. melonis 72 X. citri X. vasicola X. oryzae X. pisi X. arboricola X. hortorum species level clade 1 X. fragariae species level clade 2 X. vesicatoria X. campestris X. cucurbitae X. codiaei species level clade 3 species level clade 4 90 non pathogenic xanthomonas strains 95 species level clade 7 X. sacchari species level clade 5 species level clade 6
17 gyrb phylogeny X. euvesicatoria sp. complex 67 X. euvesicatoria sister clade (pv s dieffenbachiae, phaseoli ) X. melonis 72 X. citri X. vasicola X. oryzae X. pisi X. arboricola X. hortorum species level clade 1 X. fragariae species level clade 2 X. vesicatoria X. campestris X. cucurbitae X. codiaei species level clade 3 species level clade 4 90 non pathogenic xanthomonas strains 95 species level clade 7 X. sacchari species level clade 5 species level clade 6
18 gyrb phylogeny X. euvesicatoria sp. complex 67 X. euvesicatoria sister clade X. melonis 72 X. citri X. vasicola X. oryzae X. pisi X. arboricola X. hortorum species level clade 1 X. fragariae species level clade 2 X. vesicatoria X. campestris X. cucurbitae X. codiaei species level clade 3 species level clade 4 90 non pathogenic xanthomonas strains 95 species level clade 7 X. sacchari species level clade 5 species level clade 6
19 gyrb phylogeny X. euvesicatoria sp. complex 67 X. euvesicatoria sister clade X. melonis pv. dieffenbachiae 72 X. citri X. vasicola X. oryzae X. pisi X. arboricola X. hortorum species level clade 1 X. fragariae species level clade 2 X. vesicatoria X. campestris X. cucurbitae X. codiaei species level clade 3 species level clade 4 90 non pathogenic xanthomonas strains 95 species level clade 7 X. sacchari species level clade 5 species level clade 6
20 gyrb phylogeny X. euvesicatoria sp. complex 67 X. euvesicatoria sister clade X. melonis 72 X. citri X. vasicola X. oryzae X. pisi X. arboricola X. hortorum species level clade 1 X. fragariae species level clade 2 X. vesicatoria X. campestris X. cucurbitae X. codiaei species level clade 3 species level clade 4 90 non pathogenic xanthomonas strains 95 species level clade 7 X. sacchari species level clade 5 species level clade 6
21 Pathovar resolution gyrb Accessory gene
22 Conclusion gyrb-barcode: OK at species level : X. oryzae, X. fragariae, X. vesicatoria, X. translucens Insufficient at subspecies/pathovar level Multi-locus DNA-barcode
23 Conclusion Supplementary loci? Core genes: + phylogenetic relevance - resolution? Accessory genes: + direct link to pathovar - horizontal gene transfer - limited distribution
24 Conclusion Pathovars = special purpose classification (Provisional solution for communication, regulation and quarantine) Not covered by International Code of Names of Prokaryotes (Lapage et al., 15) Approved Lists (Skerman et al., 10) Taxonomically not well investigated Sometimes composed of multiple unrelated genotypes (biological entities?) Xanthomonas classification very dynamic DNA barcodes can provide new evidence to address complex taxonomic issues
25 QBOL, DNA barcodes to identify phytobacteria subjected to EU quarantine regulations Cottyn B., L. Detemmerman, M. Maes COST873 - Annual Meeting September 2010
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