Molecular Characterisation Of Bischofia Polycarpa Witches' Broom Phytoplasma And Its Relationship To Other Phytoplasmas In China

Java Bishopwood (Bischofia polycarpa(Levl.) Airy Shaw) is a kind of important broadleafspecies in the south of China. Bischofia polycarpa witches' broom(BiWB) disease, whosetypical symptoms includes leaves size reduction, yellowing, reddening, internode shorteningand lateral bud proliferation, occurs prevalently in its cultivation regions and leads to witches'broom, decline, dieback, even death. It is known that BiWB disease is caused by phytoplasma(previously termed mycoplasma-like organism), which was found occurring only in China sofar. Identification and systematic classification for this phytoplasma still have not been carriedout because of lacking molecular biological information. In this dissertation, molecularbiological methods were applied to identify BiWB phytoplasma isolates collected fromdifferent regions in China, and the newly classification standard were also adopted. Thephytoplasmal conserved DNAs, including 16S rRNA gene, 23S rRNA gene, ribosomalprotein(rp) gene and elongation factor EF-Tu(tuf) gene as well as 16S-23S rDNA spacerregions(SR)were amplified, cloned and sequenced. The sequence-alignments and phylogeneticanalyses of BiWB phytoplasma conserved sequences with other phytoplasmal sequencesderived from GenBank were made. The results showed that BiWB phytoplasma shared morethan 98％sequence similarity with the members of Elm yellows group in 16S rRNA gene, 99％sequence similarity with jujube witches' broom(JWB) phytoplasma in SR, 94％sequencesimilarity in 23S rRNA gene with Loofah witches' broom phytoplasma(16SrⅧ), the knownmost similar phytoplasma. BiWB phytoplasma shared 99.9％sequence similarity with JWBphytoplasma from Henan province and shared above 96％similarity with three members of16SrⅤgroup. Based on above evidences, BiWB phytoplasma has been consequently classifiedinto Elm yellows group(16SrⅤ), which shares the same group with JWB phytoplasma andcherry lethal yellows phytoplasma in China. And no mixed infection of 16SrⅥ(Cloverproliferation group) member detected previously in Bischofia polycarpa only based onPCR-RFLP analysis.Aiming at substances probably affecting PCR and obvious genomic differences betweenthis phytoplasma and Paulownia witches' broom(PaWB) phytoplasma, as well as other 16SrⅠ group phytoplasmas, the methods of DNA extraction and the conditions of PCR were studiedand modified, the optimized experimental conditions were built up, such as removal of leafmesophyll and use of in vitro cultured plantlets when extracting DNA, serial dilution oftemplate DNA and optimizing of annealing temperature during PCR amplification. And itseems that there were obvious distinction of DNA G+C content and proportion among different16Sr group phytoplasmas and various genes, which possibly exert influence upon the PCRconditions.Further molecular comparison among BiWB, PaWB, JWB, periwinkle virescence(PV)and Chinaberry witches' broom(CBWB) phytoplasmas including 23S rRNA gene sequenceanalysis and RFLP, amplification of extrachromosomal DNAs and Southern blot hybridization, as well as variation analysis of PaWB phytoplasmas collected from several regions. The resultsverified the status of BiWB phytoplasma as a member of 16SrⅤgroup and confirmed theirappropriate belongings of CBWB and PV phytoplasmas to 16SrⅠgroup. The identified folPgene of four phytoplasmas associated with folate metabolism have folate synthesis capacity, unlike clover phyllody phytoplasma that becomes pseudogenes,ΨfolP gene. This studyrevealed the molecular specialty of BiWB phytoplasma and its phylogenetic relationship andmolecular differentiation with other phytoplasmas. And the protocol for the rapid and accuratedetection, identification and differentiation of BiWB phytoplasma is outlined. Theseapproaches lay the foundation for the scientific classification and nomenclature, phytoplasmalgenomics, pathogen detection, disease quarantine, epidemiological researches and diseasemanagement.