A real-time PCR method for detection and quantification of ‘Candidatus Phytoplasma prunorum’ in its natural hosts

A real-time PCR assay conjugated with the fluorescent SYBR Green I dye has been developed for rapid, sensitive and quantitative detection of ‘Candidatus Phytoplasma prunorum’ in its natural hosts, in stone fruits such as apricot plants and in the insect vector Cacopsylla pruni. The selected primers amplified specifically a fragment 153 bp long from the rplV (rpl22) gene of ‘Ca. P. prunorum’ and not from closely related ‘Ca. P. mali’ infecting apple trees and C. picta and C. melanoneura, and ‘Ca. P. pyri’ infecting pear trees. Absolute quantification of ‘Ca. P. prunorum’ in plant and insect samples was achieved establishing two standard curves with serial dilutions of the plasmid containing ‘Ca. P. prunorum’ rpl22 target gene. The estimated concentration of phytoplasmas in infected apricot trees ranged from 1,55*10 to 6,18*10 genome units (GU) of phytoplasma DNA per nanogram of plant DNA, while in infected C. pruni ranged from 1,07*10 to 4,24*10 per tested insect.