Identification and Molecular Characterization of a Phytoplasma Associated with Pomegranate Fasciation Disease

To confirm phytoplasma infection, samples of pomegranate (Punica granatum L.) plants showing symptoms of fasciation were collected from an orchard located in Tai'an, Shandong Province, China. A fragment of approximately 1.2 kb was amplified with universal primers targeting the phytoplasma 16S rRNA gene from symptomatic pomegranate plants, while no fragment was obtained from healthy plants. The phytoplasma associated with the disease was designated as pomegranate fasciation (PoF). Two representative phytoplasma 16S rDNA gene sequences (PoF-Ch01 and PoF-Ch02) had 100% nucleotide sequence identity. The 16S rDNA sequence of PoF-Ch01 and PoF-Ch02 showed the highest similarity (99.6%) to that of 'P. granatum' phytoplasma isolate AY-PG, which belong to 16SrI-B. Further phylogenetic analysis showed that PoF-Ch01 and PoF-Ch02 belonged to a cluster of 16SrI subgroup members. In silico RFLP analysis indicated that PoF-Ch01 shared the highest similarity coefficient of 0.97 with reference strains of 16SrI-B, M and N. Actual RFLP analysis of both enzymes BstUI and BfaI confirmed that of the virtual RFLP analysis. Combining these results, we concluded that PoF was a member of the 'Candidatus Phytoplasma asteris' group (16SrI), and has very close relationship with 16SrI-B subgroup.