The identification and sequence analysis of 16S rRNA gene of Fraxinus sogdianaBunge witches’ broom phytoplasma

Abstract:

[Objective] To identify and classify the Fraxinus sogdianaBunge witches′ broom phytoplasma. [Methods] Horizontal slices of tender stem from Fraxinus sogdianaBunge were stained with aniline blue and 4,6-diamidino-2-phenylindole (DAPI), and then observed under flurescence microscopy. 16S rRNA gene sequences were amplified using direct-PCR and nested-PCR with universal primers P1/P7 and R16F2n/R16R2 for phytoplasmal 16S rRNA gene sequence. Amplicons were analyzed by restriction fragment length polymorphism (RFLP) and a phylogenetic tree was constructed based on the sequences of 16S rRNA gene. [Results] A phytoplasma was identified from Fraxinus sogdianaBunge which with symptoms of witches’ broom and was tentatively named as Fraxinus sogdianaBunge witches’ broom phytoplasma (Fraxinus sogdianaBunge WB). The 16S rRNA gene sequence was deposited in GenBank with the accession number of KF061042. Its RFLP patterns of 16S rRNA gene were the same as jujube witches′ broom phytoplasma which belong to 16Sr V-B subgroup, and it had the same phylogenetic position with jujube witches’ broom phytoplasma strain AB052876. [Conclusion] Fraxinus sogdianaBunge witches′ broom phytoplasma was identified as the member of 16Sr V-B subgroup.

Key words:Fraxinus sogdianaBunge, Phytoplasma, Aniline blue, DAPI, 16S rRNA, RFLP, Phylogenetic analysis