Molecular characterization of a new member of the 16SrV group of phytoplasma associated with Bischofia polycarpa (Levl.) Airy Shaw witches' -broom disease in China by a multiple gene-based analysis

Bischofia polycarpa (Levl.) Airy Shaw witches'-broom disease is widespread in southern China. Infected trees show symptoms of severely proliferating shoots, reduced leaf size, yellowing and reddening of leaves, and internode shortening. We detected the phytoplasma in diseased tissues by 4',6-diamidino-2-phenylindole-HCl fluorescence microscopy and polymerase chain reaction using the universal primers P1/P7. We conducted sequence similarity analyses based on 16S rRNA and 23S rRNA genes, the spacer region between the 16S and 23S rRNA genes (ISR), the partial ribosomal protein-encoding operon (rp), and the elongation factor Tu (tuf) gene of the BiWB phytoplasma. These analyses unanimously showed that the BiWB phytoplasma belongs to the elm yellows group (the 16SrV group). Phylogenetic and computer-simulated restriction fragment length polymorphism analyses using the nearly full-length 16S rRNA gene sequence demonstrated that the BiWB phytoplasma is more closely related to the subgroup 16SrV-B than to the other subgroups in 16SrV. It may represent a novel subgroup, designed as 16SrV-H, which is distinct from other subgroups of phytoplasmas in the 16SrV group.