Rapid screening for phytoplasma presence in flower crops using tuf gene barcode

Several molecular markers are currently available for phytoplasma strain discrimination. However, these markers often cannot be used for identification of phytoplasmas belonging to different ribosomal groups, or are not suitable for routine diagnostics. The DNA barcode amplicon based on the elongation factor Tu (tuf) gene for universal phytoplasma identification (420-444 bp) was employed for verification of phytoplasma presence in samples from different plant species in PCR/RFLP analyses. Samples from 13 flower species showing symptoms suggesting phytoplasma presence and from corresponding asymptomatic plants were tested. The symptomatology present in the tested samples ranged from virescence in orchid, narcissus, Centaurium erythraea, primula, gladiolus, surphinia and hydrangea, to phyllody and/or flower malformation in ranunculus, carnation, petunia, statice, helicrysum, and gerbera. PCR amplicons of the expected size were obtained from all symptomatic samples. No amplicons were obtained from symptomless plants of the same species or negative controls devoid of DNA. The RFLP analyses carried out with TruI, Tsp509I, TaqI restriction enzymes allowed the differentiation among phytoplasmas in 3% agarose gels and was useful for rapid screening of large sample numbers. The phytoplasma differentiation achieved is in agreement with published phytoplasma groupings based on 16S rDNA. In case of phytoplasmas relevant for quarantine, sequencing may be necessary for confirmation. Tuf reference barcodes are deposited in the NCBI GenBank and in the Q-bank (http://www.qbank. eu/Phytoplasmas/), a freely available online identification tool for plant pests and pathogens of quarantine status.