PCR indexing of phytoplasma-infected micropropagated periwinkle treated with PAP-II, A ribosome inactivating protein from Phytolacca Americana leaves

To verify the possibility of phytoplasma elimination from tissue cultures without employing meristem-tip culture or antibiotics, micropropagated periwinkle shoots infected with an aster yellows phytoplasma (Hydrangea virescence phytoplasma: HyV) were obtained. Preliminary tests carried out on shoots immersed in sterile water containing decreasing concentrations of PAP-II, for different periods of time showed an increased number of necrotic shoots after 48 hours exposure. Similar tests showed that when PAP-II was added to the medium by percolation no phytotoxicity was detectable. Batches of 1-3 cm long shoots grown in a solid medium were treated with serial dilutions of purified PAP-II. After periods of growth varying from 15 to 150 days, shoots ranging from 0.1 to 0.6 g were used for DNA extraction with a silica gel system. Nested-PCR employing general and group 16SrI specific phytoplasma primers [R16F2n/R2 followed by R16(I)F1/R1] was performed to verify phytoplasma elimination in a total of about 200 infected shoots. The percentage of phytoplasma-free samples ranged from 40 to 50 % in dilutions ranging from 1:10 to 1:1,000 and for periods of time between 50 and 150 days. Asymptomatic shoots deriving from HyV infected material were found to be positive in nested-PCR as well as the symptomatic ones. Periwinkle infected shoots maintained in micropropagation on the same medium as control without PAP-II showed phytoplasma presence when tested in the same nested-PCR system.