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

Several molecular markers are nowadays available for phytoplasma strains discrimination, however, they often cannot be used for identification of phytoplasmas belonging to different ribosomal groups or are not friendly 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 referable to phytoplasma presence and from corresponding asymptomatic plants were tested. The symptomatology present in the tested samples ranged from virescence in orchid, narcissus, gentian, primula, gladiolus, surphinia and hydrangea to phyllody and or flower malformation in ranunculus, carnation, petunia, statice, helicrysum, and gerbera. PCR products of the expected length were obtained from all symptomatic samples and no amplicons were produced from negative controls devoid of DNA and from healthy plants of the same species. The RFLP analyses carried out with TruI, Tsp509I, TaqI restriction enzymes allowed the differentiation among phytoplasmas in agarose 3% gels and resulted useful for fast screening of large number of samples. The achieved phytoplasma differentiation is in agreement with published phytoplasma groupings based on 16S rDNA. The visualization of restriction profiles in agarose is a handily tool for large number of sample processing enclosing identification ability. 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 newly developed Q-bank (http://www.q-bank.eu/Phytoplasmas/), a freely available online identification tool for plant pests and pathogens of quarantine status.