DNA barcoding of phytoplasmas

Phytoplasmas infect a high number of plant species. Based on the 16S ribosomal gene sequence, over 20 RFLP groups have been established within the ‘Candidatus Phytoplasma’ taxon, some of which are of quarantine status. To date, no protocol for in vitro cultivation of phytoplasma has been developed, making classical microbiological identification procedures impossible. The basic phytoplasma identification methods is based on RFLP analysis of a 1.2 kbp region of the 16S rDNA, however, this method is not easy to set up and it only considers a part of the available phytoplasma sequence information. It has been suggested that universally amplified, short, and highly variable DNA barcodes may help to rapidly identify organisms. As part of QBOL project a universal DNA barcoding based tool for phytoplasma identification was developed. Two sets of primers amplifying a fragment of the Tuf gene and a fragment of the 16S rDNA gene, respectively, were designed and the potential of these fragments as DNA barcodes was verified. Successful amplification and sequencing of more than 150 phytoplasma strains, and ability to separate various phytoplasma strains to ‘Candidatus species’ level, 16S ribosomal group and sub-group level suggest that these barcodes are efficient phytoplasma identification tools