QBOL - Identification of phytoplasmas using DNA barcodes

Climate change, expansion of the EU and the increase of international trade may facilitate the spread of phytoplasma associated diseases, therefore a quick and handily system for correct plant pathogen identification is in great demand. DNA barcoding has arisen as a robust and standardised approach to species identification. QBOL, a new project funded by EU FP7 aims to create a barcode database for all quarantine plant pathogens and to make it available for plant health diagnosticians. Phytoplasma ‘barcoding’ has been performed for many years, particularly using the 16S rDNA, but also other genes such as secY, secA, tuf and ribosomal proteins; however most of these regions span more than 1 kb and/or primers are not generic, which make them impractical for routine barcoding of unknown phytoplasmas. Available sequences of elongation factor Tu (Tuf) and 16S genes were explored for selecting regions suitable for phytoplasma DNA barcoding to develop robust markers of a size that can easily be sequenced (400-600 bp) and that can be obtained from most, if not all phytoplasma ribosomal groups and/or ‘Candidatus Phytoplasma’ species using generic primers. A number of phytoplasma strains maintained in periwinkle and field collected were used for PCR amplification with newly developed primers for Tuf and 16S regions and then sequenced. The 5’ end of the Tuf and the 5’ end of 16S genes were used for barcoding. Sequences of approximately 450 bp for Tuf and 625 bp for the 16S gene were obtained from 60 and 40 phytoplasma strains respectively, belonging to 10 different 16Sr groups. Using these sequences as barcodes it was possible to identify the phytoplasmas into ‘Candidatus species’ or 16Sr groups. The obtained sequences will be available in the newly developed QBOL database.