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A Barcode Database to Identify the Vectors of Xylella Fastidiosa in Europe

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A Barcode Database to Identify the Vectors of Xylella Fastidiosa in Europe A barcode database to identify the vectors of Xylella fastidiosa in Europe Jean-Claude Streito, Eric Pierre, Guénaëlle Genson, Maxime Bellifa, Marguerite Chartois, Jean-François Germain, Astrid Cruaud, Jean Yves Rasplus To cite this version: Jean-Claude Streito, Eric Pierre, Guénaëlle Genson, Maxime Bellifa, Marguerite Chartois, et al.. A barcode database to identify the vectors of Xylella fastidiosa in Europe. 2. European conference on Xylella fastidiosa: how research can support solutions, Oct 2019, Corse, France. hal-02790440 HAL Id: hal-02790440 https://hal.inrae.fr/hal-02790440 Submitted on 5 Jun 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. A barcode database to identity the vectors of Xylella fastidiosa in Europe. By Streito Jean-Claude, Pierre Eric, Genson Guénaëlle, Bellifa Maxime, Chartois Marguerite, Ajaccio 29/10/2019 †Germain Jean-François, Astrid Cruaud & Jean-Yves Rasplus. Presentation plan Introduction : why bacode Xylella’s vectors? Material & methods: how to produce reliable barcodes? Results and taxonomic uncertainties Conclusion .02 Streito et al. / Barcoding of Xylella fastidiosa vectors Ajaccio 29/10/2019 _01 Introduction Why bacode Xyella’s vectors ? .03 Streito et al. / Barcoding of Xylella fastidiosa vectors Ajaccio 29/10/2019 Why barcode Xylella's vectors ? 1) Potential vectors of Xf are diversified 4 Hemiptera famillies, more about 120 spp. for EU Aphrophoridae (29 spp.) Cercopidae (7 spp.) Cicadellidae (9 spp.) Cicadidae (79 spp.) For an overview of Xf pupative vectors see the poster P58 by Wilson et al. .04 Streito et al. / Barcoding of Xylella fastidiosa vectors Ajaccio 29/10/2019 Why barcode Xylella's vectors ? 2) Main vectors must be dissected to be identified It is possible and reliable but time-consuming and not suitable for mass screening of Xf in insects .05 Streito et al. / Barcoding of Xylella fastidiosa vectors Ajaccio 29/10/2019 Why barcode Xylella's vectors ? .06 Streito et al. / Barcoding of Xylella fastidiosa vectors Ajaccio 29/10/2019 Why barcode Xylella's vectors ? 1) Potential vectors of Xf are diversified 2) Main vectors must be dissected to be identified 3) The taxonomy of Xf’s vectors is not 100% satisfactory Barcoding is known to be adequate to check for taxonomical problems: cryptic species and synonymies It is why we started to barcode the vectors of Xf since its discovery in Corsica in 2015 .07 Streito et al. / Barcoding of Xylella fastidiosa vectors Ajaccio 29/10/2019 _02 Materials & Methods .08 Streito et al. / Barcoding of Xylella fastidiosa vectors Ajaccio 29/10/2019 Sampling : why ? 1) Collect as many species as possible 2) Get fresh material containing high quality DNA (specimens from collections contain damaged DNA and have high- value: not possible to squeeze them) 3) Access to abundant material for the development of XF detection techniques .09 Streito et al. / Barcoding of Xylella fastidiosa vectors Ajaccio 29/10/2019 Sampling : How and Where ? 1) Sweeping and beating 2) Two periods : - 2009-2012 non European vectors (in the framwork of Qbol project) - After August 2015 European vectors North American vectors European vectors Xylella samples QBOL samples .010 Streito et al. / Barcoding of Xylella fastidiosa vectors Ajaccio 29/10/2019 Barcoding methods: multiple quality controls Species to be Sampling conditioning 1) IdentificationIdentification by Databases included storage a taxonomist Web portal Philaenus italosignus Philaenus lukasi Philaenus maghresignus Philaenus signatus Philaenus spumarius Philaenus tarifa Philaenus tesselatus 2)DNA Non extraction destructive and Collections extractionstorage ADN 3) Vouchers PCR amplification COI Sequencing Phylogenetic analysis 1st quality control: sequences 2nd quality control .011 Streito et al. / Barcoding of Xylella fastidiosa vectors Ajaccio 29/10/2019 Non destructive DNA extraction One important step: that allows vouchering and morphological re-examination in case of mismatch between morpho and barcodes Extraction Voucher Observation .012 Streito et al. / Barcoding of Xylella fastidiosa vectors Ajaccio 29/10/2019 Barcoding methods: Standard Barcode: COI 656 bp (Herbert et al., 2003) First: Classical PCR and Sanger sequencing Now: two-step PCR and Miseq sequencing .013 Streito et al. / Barcoding of Xylella fastidiosa vectors Ajaccio 29/10/2019 Barcoding methods: multiple quality controls Species to be Sampling conditioning 1) IdentificationIdentification by Databases included storage a taxonomist Web portal Philaenus italosignus Philaenus lukasi Philaenus maghresignus Philaenus signatus Philaenus spumarius Philaenus tarifa Philaenus tesselatus 5) Consistency 2)DNA Non extraction destructive and Collections extractionstorage ADN morpho/molecular 3) Vouchers identification PCR amplification COI Sequencing 4) Pipeline to check Phylogenetic analysis 1st quality control: sequences sequences (non‐coding, 2nd quality control contaminants, numts…) .014 Streito et al. / Barcoding of Xylella fastidiosa vectors Ajaccio 29/10/2019 Photography of specimens Help for morphological identification Male specimens were dissected Images of both habitus and genitalia were produced with a Keyence VHX5000 Microscope Photos in natura have also been taken .015 Streito et al. / Barcoding of Xylella fastidiosa vectors Ajaccio 29/10/2019 Database : validated sequences and photos Sequences are stored in Database Arthemis http://arthemisdb.supagro.inra.fr/DefaultInfo.aspx?Page=Home Freely available in Arthemis : -Pictures - Sequences validated Arthemis DB@se focuses on pests and their natural enemies. To date it contains 21 169 COI sequences of Arthropods .016 Streito et al. / Barcoding of Xylella fastidiosa vectors Ajaccio 29/10/2019 _03 Results .017 Streito et al. / Barcoding of Xylella fastidiosa vectors Ajaccio 29/10/2019 Sampling North American vectors European vectors Total putative vectors : 22 849 - Aphrophoridae : 11 199 - Cercopidae : 165 Xylella samples - Cicadellidae : 11 364 QBOL samples - Cicadidae : 121 .018 Streito et al. / Barcoding of Xylella fastidiosa vectors Ajaccio 29/10/2019 Number of barcodes and pictures Number of Xf vectors included: http://arthemisdb.supagro.inra.fr/DefaultInfo.aspx?Page=Home European vectors: 21(+16) spp. 234(+151) COI Non European vectors: 23 spp. 173 COI Total number of spp. 44(+32) 407(+151) COI Number of pictures : 271 (24 European spp. and 5 non European spp.) .019 Streito et al. / Barcoding of Xylella fastidiosa vectors Ajaccio 29/10/2019 Results: Philaenus Number of Bold 7 spp. in Europe Espèces / sous‐espèces Pictures Séquences GenBank (in process) 5 spp. sequenced and Philaenus italosignus 74 1pictured Philaenus loukasi 00 1 Philaenus maghresignus 91 2+ Philaenus signatus (7) (7) 1 few sequences available Philaenus spumarius 32 39(11) > 100 in international db Philaenus tarifa 00 2 Philaenus tesselatus 11 29 (8) 2 Philaenus arslani (Liban) 0 0 1 All European spp. Philaenus elbursianus (Iran) 0 0 0 Philaenus iranicus (Iran) 0 0 0 sequenced .020 Streito et al. / Barcoding of Xylella fastidiosa vectors Ajaccio 29/10/2019 Results: taxonomic concerns Philaenus COI separates all spp. studied except spumarius/tesselatus .021 Streito et al. / Barcoding of Xylella fastidiosa vectors Ajaccio 29/10/2019 Results: taxonomic concerns spumarius/tesselatus Philaenus Philaenus spp. : ‐ same variable habitus ‐ aedeagus diagnostic but difficult between spumarius/tesselatus but Is P. tesselatus a good species ? For more details see the poster P60 by Seabra et al. !! .022 Streito et al. / Barcoding of Xylella fastidiosa vectors Ajaccio 29/10/2019 Results Neophilaenus Number of Bold 11 spp. in Europe Espèces / sous‐espèces Pictures Séquences GenBank (in process) 2 spp. sequenced Neophilaenus albipennis 2(1)05 pictured Neophilaenus angustipennis 00 0 Neophilaenus campestris 931(8)3+ Neophilaenus exclamationis 9(1)0Nearly no Neophilaenus infumatus 000barcodes Neophilaenus limpidus 000 Neophilaenus lineatus 9 8 (6) >100 in international db Neophilaenus longiceps 00 0 Neophilaenus minor 2(2)0 Neophilaenus modestus 00 0<50% European Neophilaenus pallidus 00 0spp. sequenced .023 Streito et al. / Barcoding of Xylella fastidiosa vectors Ajaccio 29/10/2019 Results: taxonomic concerns Neophilaenus Morphology is not totaly clear Number of Bold Espèces / sous‐espèces Pictures Séquences GenBank (in process) Neophilaenus albipennis 2(1)0 Neophilaenus angustipennis 00 0 Neophilaenus campestris 931(8)3 Neophilaenus exclamationis 9(1)0 Neophilaenus infumatusA difficult genus,000 more Neophilaenus limpidusbarcodes are 000needed Neophilaenus lineatus 9 8 (6) >100 Neophilaenus longiceps 00 0 Neophilaenus minor 2(2)0 Neophilaenus modestus 00 0 Neophilaenus pallidus 00 0 .024 Streito et al. / Barcoding of Xylella fastidiosa vectors Ajaccio 29/10/2019 Results Aphrophora 7 spp. in Europe Number of Bold Espèces / sous‐espèces Pictures Séquences 4 spp. sequenced GenBank (in process) and pictures Aphrophora alni 7 11 (6) >100 Aphrophora corticea 95 (5) 3 Aphrophora major 00 0In international db Aphrophora pectoralis 13 3 1 confusion between
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