Molecular Identification Of Phytoplasmas Associated With Stem Fasciation Of Chinese Scholar Tree And Crape Myrtle

Chinese scholar tree and Crape myrtle are two main ornamental trees. Due to theoccurrence of stem fasciation, characterised by enlargement and flattening of stems andexcessive proliferation of shoots, eventually die. This will caused large economic losses togarden producers. This study used molecular biology techniques to identification of theoriginal body of Chinese scholar tree and crape myrtle disease.The main findings are as follow as:1. We collected stem with symptoms of stem fasciation,16SrRNA gene was amplified usingphytoplasma-universal16S rDNA primer pairs P1/P7followed by nested PCR with primer pairR16F2n/R16R216SrRNA gene was cloned and sequenced. With a comparison of nucleotide sequencehomology between it and the represent strains in subgroups of16SrI, it demonstrated that thephytoplasmas are related to strain AY265206（16SrI-D）, the virtual RFLP analysis showed their RFLPpattern were same to the represent strains M30790and NC₀05303in16SrI-B, and the phylogenetic treesuggested they belong to16SrI-D. According to rhe results, we classified the phytoplasmas into16SrI-（B）D.2. Rp gene of the phytoplasma associated with Chinese scholar tree was cloned andused for sequence homology，amono acid homology and phylogenetic analysis. Accordingto the results, it related to PaWB in Qujing Yunnan Province, and clustered together withPaWB, these indicated the rp was a member of16SrI-D/rpI-D.3. Tuf gene was also cloned and used for homology and phylogenetic analysis, as itsgenetic relationship was related to tufI-B, we classified tuf gene of Chinese scholar treestem stasfication into tufI-B.4. In this paper, we report a modified DNA extraction procedure, based on thehexadecyltrimethylammonium bromide （CTAB） method, which adds polyvinyl pyrrolidone（PVP） powder before grinding plant tissues. The results showed that both polyphenols andpolysaccharides in crape myrtle tissues were removed by adding PVP powder in anappropriate ratio （PVP powder/plant tissue, w/w） and the optimal ratios of PVP powder/plant tissues were2:10and1:10for stems and leaves, respectively. In the presentstudy, an original protocol method which added PVP powder when grinding was developedfor high-quality DNA isolation from crape myrtle and the extracted DNA could be used forPCR, RAPD-PCR and16S rDNA amplification of phytoplasmas.We classified the phytoplasma in this research into ’Candidatus Phytoplasma asteris’,the phytoplasma associated with Chinese scholar tree stem fasciation was into16SrI-（B）D/rpI-D/TufI-B, and the phytoplasma associated with crape myrtle stem fasciationwas into16SrI-（B）D. It’s the first report that16SrI-（B）D infected Chinese scholar tree andcrape myrtle.