Standardization of PCR technique for detection of phytoplasmas associated with sandal spike and stachytarpheta phyllody and RFLP analysis of 16S rDNA genome

Sandal (Santalum album L.) is a valuable tree of southern India. It is severely affected by spike disease, which is characterized by witches broom type symptoms, Spike disease is caused by a phytoplasma with its uneven distribution in the phloem tissues. PCR technique for detection of Sandal spike and Stachytarpheta phyllody was standardized using gradient PCR, PCR product of approximately 1.8k of 16Sr DNA consistently amplified with the universal phytoplasma specific primer pairs of P1/P7. Nested PCR technique performed in order to confirm the association of phytoplasma with the Sandal spike and Stachytarpheta phyllody resulted in the amplification of product of 1.2kb of 16Sr DNA obtained using primer pair R16F2n/R16R2 with the 1.8kb PCR product was used as template. Distinct RFLP pattern was observed between Sandal spike phytoplasma and Stachytarpherta phyllody phytoplasma when these are digested with AluI and HinfI endonuclease enzyme. This clearly revealed that Sandal spike phytoplasma was different from Stachytarpheta phyllody phytoplasma.