Bulletin OEPP/EPPO Bulletin (2021) 51 (1), 100–143 ISSN 0250-8052. DOI: 10.1111/epp.12724 European and Mediterranean Plant Protection Organization Organisation Europe´enne et Me´diterrane´enne pour la Protection des Plantes PM 7/129 (2) Diagnostics PM 7/129 (2) DNA barcoding as an identification tool for a number of regulated pests Specific scope Specific approval and amendment This Standard describes the use of DNA barcoding proto- First approved in 2016–09. cols in support of identification of a number of regulated Revised in 2020–10. pests and invasive plant species comparing DNA barcode regions with those deposited in publicly available sequence databases.1 It should be used in conjunction with PM 7/76 Use of EPPO diagnostic protocols. identification at the required taxonomic level in several pest 1. Introduction groups. DNA barcoding is a generic diagnostic method that uses a DNA barcoding protocols for eukaryotes and prokaryotes short standardized genetic marker in an organism’s DNA to (a novelty in the DNA barcoding field) were developed and aid identification at a certain taxonomic level. The chosen validated within the Quarantine Organisms Barcoding of marker region should reflect the group taxonomy of the tar- Life (QBOL) Project financed by the 7th Framework Pro- get species. Therefore, the marker region should provide a gramme of the European Union. Within the DNA barcoding high interspecific variability and low intraspecific differ- EUPHRESCO II project, test protocols for several quaran- ences, and should enable the identification of as many spe- tine pests and invasive plant species were added, and the cies as possible belonging to a shared higher taxonomical use of polymerases with proofreading abilities was intro- level such as genus, family or order (e.g. Chen et al., duced to minimize the risk of polymerase chain reaction 2013). An organism is identified by finding the closest (PCR) errors. In addition, amplification primers were M13- matching reference record. The first genetic marker to be tailed when possible to improve the user-friendliness of the described as a ‘barcode’ was the mitochondrial cytochrome protocols, allowing the generation of sequence data with a c oxidase I (COI) gene, which is used for species identifica- minimum number of sequencing primers. Additional proto- tion in the animal kingdom (Hebert et al., 2003). Later the cols have been added in the second version of the Standard chloroplast large subunit ribulose-1,5-bisphosphate carboxy- based on national projects conducted at Austrian Agency lase-oxygenase (rbcL) gene (Hollingsworth et al., 2009) for Health and Food Safety (AGES, AT), Flanders Research and the nuclear ribosomal internal transcribed spacer (ITS) Institute for Agriculture, Fisheries and Food (ILVO, BE) region (Schoch et al., 2012) were proposed as barcodes for and Netherlands Food and Consumer Product Safety the plant and fungi kingdoms, respectively. Authority (NVWA, (NL). The use of a single barcode region does not provide suf- Regulated organisms are identified by finding the closest ficient reliability for the identification of the majority of matching reference record using a combination of Basic regulated pests. Therefore, several short standardized Local Alignment Search Tool (BLAST) hit identity, multi- genetic markers have been identified as ‘barcodes’ for locus sequence analysis (MLSA) and clustering in species- specific clades using multiple databases containing sequence data of regulated organisms and related species. 1Use of brand names of chemicals or equipment in these EPPO Stan- Pest species in this Standard were selected on the basis of dards implies no approval of them to the exclusion of others that may their pest status, economic impact, availability of material also be suitable. 100 ª 2021 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 51, 100–143 PM 7/129 (2) DNA barcoding 101 and pre-existing knowledge of loci with sufficient resolu- Ralstonia solanacearum species complex (Appendix 2) and tion. test 2.8 TEF1 (Appendix 3)] can be monitored using a sin- This EPPO Standard describes the DNA barcoding proto- gle control. When amplified, the synthetic PACs yield cols developed for the identification of a number of regu- amplicons ranging from 560 to 720 base pairs, depending lated arthropods, bacteria, fungi and oomycetes, invasive on the primers used. When sequenced, the synthetic PACs plant species, nematodes and phytoplasmas. Each organism can easily be identified since, after translation of the nucleic group is covered in a separate appendix. Protocols describe acid sequence (reading frame 1, standard code), the follow- the extraction of nucleic acids and the amplification of ing amino acid sequence is obtained twice: *KEEP*- short standardized marker(s). Since the identification of reg- CALM*THIS*IS*MERELY*A*VERY*STRANGE*RE- ulated pests is often based on several different markers, FERENCE*PHRASE*WITH*EIGHTY*FIVE*CHARAC- diagnostic schemes are provided to aid the selection of TERS (stop codons are indicated as *). Synthetic PAC appropriate protocols. When more than one marker is nec- sequences are presented in Appendix 9 and are available essary, the markers are either used in parallel for species from the NCBI: PAC arthropods v.1 (KT429638), PAC identification (e.g. invasive plant species and phytoplasmas) bacteria v.1 (KT429643), PAC fungi v.1 (KT429642), PAC or a single marker is first used for genus identification (e.g. invasive plant species v.1 (KT429639), PAC nematodes v.1 16S rDNA for bacteria) and, depending on the genus, a sec- (KT429641) and PAC phytoplasmas v.1 (KT429640), and ond marker (sometimes in parallel with a third marker) is can be ordered from commercial companies producing syn- used for identification to species level. For some thetic genes or gBlocks (e.g. ThermoFisher, IDT, Bioma- Xanthomonas bacteria a third marker is needed for identifi- tik). cation at the pathovar level. For each identification based on several markers all consensus sequences produced need 3. Feedback on this Diagnostic Protocol to be analysed in an MLSA, which can be done in EPPO- Q-bank (https://qbank.eppo.int/). The generation of If you have any feedback concerning this Diagnostic Proto- sequence data, assembly of raw sequence data and analysis col, or any of the tests included, or if you can provide addi- of consensus sequences using BLAST and MLSA in online tional validation data for tests included in this protocol that databases is discussed in Appendix 7. Appendix 8 provides you wish to share please contact [email protected]. an example of a sequencing analysis report that can be used to collate all relevant data and Appendix 9 provides infor- 4. Protocol revision mation on synthetic positive amplification controls (PACs). It should be noted that the outcome of DNA barcoding An annual review process is in place to identify the need tests can be negatively affected by the incompleteness of for revision of Diagnostic Protocols. Protocols identified as databases, incorrectly identified species in databases, the needing revision are marked as such on the EPPO website. amplification of pseudogenes or nuclear mitochondrial When errata and corrigenda are in press, this will also be DNAs (NUMTs) and introgression or hybridization events. marked on the website. For that reason, the analysis of sequence data should be performed by proficient operators. DNA barcoding is conse- Acknowledgements quently used in support of identification at a certain taxo- nomic level. Origin, host plant and other characteristics This protocol was originally drafted by BTLH van de (e.g. morphological, biochemical, reactions on indicator Vossenberg, M Westenberg and M Botermans, Dutch plants) are typically needed to complete the diagnosis. National Plant Protection Organization, PO Box 9102, 6700 Video tutorials of barcoding training sessions produced in HC Wageningen, the Netherlands, J Hodgetts, Fera Science the framework of the PRACTIBAR Euphresco project (Gio- Limited, Sand Hutton, York YO41 1LZ, UK, and B Cottyn, vani et al., 2020) are available on YouTube (https://www. Institute for Agricultural and Fisheries Research, Plant youtube.com/playlist?list=PLoVf4Pt04Db53pUVTI8qwc Sciences Unit, Crop Protection, Burgemeester van Gans- WkWgUgg46gm). berghelaan 96, bus 2, 9820, Merelbeke, Belgium. The first revision was drafted by T. Warbroek, Dutch National Plant Protection Organization, PO Box 9102, 6700 HC Wagenin- 2. Reference material gen, the Netherlands, B Cottyn and J van Vaerenbergh, A single synthetic PAC per organism group can be used to Institute for Agricultural and Fisheries Research, Plant assess the efficiency of the PCR amplification. It can also Sciences Unit, Burgemeester van Gansberghelaan 96, 9820, be used as a standardized process control from amplifica- Merelbeke, Belgium, and R Gottsberger, Austrian Agency tion until sequence analysis and will give insight into the for Health and Food Safety (AGES), Department for repeatability and reproducibility of each test (see also Molecular Diagnostics of Plant Diseases, Spargelfeldstr. Appendix 7, section 5.2). The synthetic PACs presented in 191, 1220 Vienna. It was reviewed by the EPPO Panel on Appendix 9 were designed in such a way that all tests in Diagnostics and Quality Assurance. Most of the DNA bar- one appendix [except test 2.4 Conventional PCR egl coding protocols in this standard were developed, optimized ª 2021 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 51, 100–143 102 Diagnostics and validated in an international test performance study simple adaptations. Molecular Phylogenetics and Evolution 48, 758– within the QBOL Project financed by the 7th Framework 763. Program of the European Union and the DNA Barcoding Holterman M, van der Wurff A, Van den Elsen S, Van Megen H, Bongers T, Holovachov O et al. (2006) Phylum-wide analysis of EUPHRESCO II Project. SSU rDNA reveals deep phylogenetic relationships among nematodes and accelerated evolution toward crown clades. Molecular References Biology and Evolution 23, 1792–1800. Hu M, Hoglund J, Chilton NB, Zhu XQ & Gasser RB (2002) Mutation Benson DA, Cavanaugh M, Clark K, Karsch-Mizrachi I, Lipman DJ, scanning analysis of mitochondria cytochrome c oxidase subunit 1 Ostell J et al.