Preliminary results of axenic growth of phytoplasmas from micropropagated infected periwinkle shoots

Periwinkle shoots infected with stolbur phytoplasma strain CH1 (group 16SrXII), dodder transmitted from grapevine infected with “bois noir” disease in Italy, were employed for phytoplasma growth trials in axenic medium. Sterile 1-2 mm stems from two different shoots of two years in vitro growing cultures, were cut longitudinally with razor blades under sterile conditions, immersed in 2 tubes each containing 2 ml Mycoplasma Experience liquid medium (a medium suitable for a wide range of mycoplasmas containing pig serum, a broth base and yeast extract) and maintained in an incubator at 26°C for 7 days. About 300 µl of the medium containing the cut shoots were then transferred to new tubes containing 2 ml of fresh medium. Twenty days after the transfer, PCR assays were carried out using 1 µl medium, denaturated by boiling, as template. The R16F2/R2 primers specific for phytoplasma 16S ribosomal gene were employed for 35 cycles at 47°C annealing temperature. RFLP analyses with TruI carried out on the 4 amplicons of the expected 1.2 kb length obtained with the 4 tubes inoculated showed the presence of 16SrXII phytoplasma DNA. The tubes of the first transfer were then incubated under the same conditions for 9 months when acid colour-changes were seen. Aliquots of 100 µl from the 4 tubes were transferred to 4 tubes of fresh medium which all gave acid colour changes after incubation for 3 days, but PCR testing for phytoplasmas was negative. However, after 10 days’ incubation, PCR testing was positive for the presence of phytoplasma DNA. PCR amplification was carried out using as template 1 µl of the pellets obtained from full speed centrifugation of 100 µl medium, resuspended in 10 µl of sterile distilled water. Primers employed for these latter amplifications were R16(I)F1/R1, specific for phytoplasma groups 16SrI, II and XII, and were used under published conditions; RFLP analyses confirmed that 16SrXII phytoplasmas were present in the broth media after transfers. Amplification of about 600 bp in the 16Sr of phytoplasmas followed by direct sequencing confirmed RFLP results. Further experiments are in progress.