Identification of phytoplasmas using DNA barcodes of selected genes.

Barcode regions are used to identify living organisms and the requirements are: polymorphisms to discriminate close relatives; conserved regions for primer design; ideally short 500-700 bp regions. Phytoplasma identification is carried out in a new project funded by EU FP7 to generate barcode sequences from a selected set of genetic regions and for the relevant/quarantine phytoplasmas listed below. – Elm phloem necrosis (ribosomal subgroup 16SrV-A, strain EY) – Peach rosette (ribosomal group 16SrIII) – Peach X (ribosomal subgroup 16SrIII-A, strains CX and WX) – Peach yellows (ribosomal group 16SrIII) – Strawberry witches’ broom (ribosomal subgroup 16SrI-C) – Apple proliferation ’Candidatus Phytoplasma mali’ (ribosomal subgroup 16SrX-A, strains AP, AT) – Apricot chlorotic leafroll ’Candidatus Phytoplasma prunorum’ (rib. subgroup 16SrX-B, ESFY) – Pear decline ’Candidatus Phytoplasma pyri’ (ribosomal subgroup 16SrX-C, PD) – Palm lethal yellowing (ribosomal group 16SrIV) – ’Witches broom’ on Citrus ’Candidatus Phytoplasma aurantifolia’ (ribosomal subgroup 16SrII-B) – Grapevine flavescence doreé (ribosomal subgroups 16SrV-C and 16SrV-D) – Potato stolbur (ribosomal group 16SrXII) – Potato purple top wilt (ribosomal groups 16SrI, 16SrVI, 16SrXVIIII) 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 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. A number of phytoplasma strains (about 60) 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 12 different 16Sr groups. Using these sequences as barcodes it was possible to identify the phytoplasmas into ‘Candidatus species’ or into 12 of the described 16Sr groups.