Real-time PCR for simultaneous and quantitative detection of quarantine phytoplasmas from apple proliferation (16SrX) group

A real time PCR assay conjugated with the fluorescent SYBR® Green I dye has been developed for rapid, sensitive and quantitative detection of 'Ca. Phytoplasma pyri', 'Ca. P. prunorum' and 'Ca. P. mali', quarantine members of apple proliferation (16SrX) group. The selected primers amplify specifically a target of 217-bp fragment from the 16Sr gene region of the 16SrX group and not from any other tested phytoplasma groups. An artificial template consisting in a plasmid clone of a 1785-bp DNA fragment of the 16S rRNA gene, 16S/23S rDNA spacer region, tRNA-Ile and partial 23S rRNA gene of a 'Ca. P. prunorum' isolate, was used to establish a calibration curve to evaluate the number of amplified targets per sample. The sensitivity of the technique was similar to nested-PCR (10 copies of the amplified target per μl). The estimated concentration of phytoplasmas in infected pear, plum and apricot trees ranged from 9.7×103 to 3.0×105 phytoplasmas per gram of tissue. The method offers the possibility to detect simultaneously, in a single reaction, all quarantine phytoplasmas affecting fruit trees hosts in Europe.