Detection And Identification Of Six Phytoplasmal Diseases In Xinjiang

Jujube witches’ broom of Xinjiang, Sophora alopecuroides witches’ broom, FraxinussogdianaBunge witches’ broom, Phragmites australis witches’ broom, Elm witches’broom andXanthoceras sorbifolia Bunge witches’ broom were detected and identified through molecular biologymethods in this study. Another approaches to determine whether diseased plants harbouredphytoplasma, horizontal slices of tender stem from Fraxinus sogdianaBunge and elm with and withoutsymptoms were stained with aniline blue and4,6-diamidino-2-phenylindole (DAPI), and then observedunder flurescence microscopy.16S rRNA gene sequences were amplified using direct-PCR and nested-PCR methods with universalprimers P1/P7and R16F2n/R16R2for phytoplasmal16S rRNA gene sequence from the six kinds of plants.DNA fragments of1.2kb were amplified from the total DNA of plants that showing symptoms. After blastcomparisons and a dendrogram (MEGA5.0, USA) was constructed by Neighbour-Joining method, theresults showed that the six phytoplasmas were identified as the member of16Sr V-B subgroup. Restrictionfragment length polymorphism showed that the patterns of actual restriction were the same as virtualrestriction, and the six phytoplasmas were members of16Sr V-B subgroup, the results were consistent withthat of phylogenetic analysis.Tuf gene sequences were amplified with universal primers fTufu／rTufu for phytoplasmal tufgene sequence from five kinds of plants (DNA fragment of tuf gene was not amplified fromXanthoceras sorbifolia Bunge witches’ broom phytoplasma). After nucleotide homology analysis, adendrogram was constructed by Neighbour-Joining method, the results showed that tuf gene sequencesfrom the five kinds of plants shared most similarity with Jujube witches’ broom phytoplasmas whichwere members of16Sr V-B subgroup. The results were consistent with that of the analysis results of16S rRNA gene.16S-23S rRNA spacer region sequences were amplified using direct-PCR and nested-PCRmethods with primers P1/P7and R16F2n/R16R2. DNA fragments of0.36kb were amplified from thetotal DNA of Jujube plants and Sophora alopecuroides plants that showing witches’ broom symptoms,about0.4kb DNA fragments were amplified from Fraxinus sogdianaBunge witches’ broomphytoplasma, Phragmites australis witches’ broom phytoplasma, Elm witches’broom phytoplasma,and Xanthoceras sorbifolia Bunge witches’ broom phytoplasma. Comparison of nucleotide homologyof the six phytoplasmas’16S-23S rRNA spacer region showed that the homology were94.1-99.4％, itshowed that the six phytoplasma diseases can be caused by different phytoplasma strains, they wereidentified as different strains of16Sr V–B subgroup.Specific fluorescence zone could be seen within horizontal slices of tender stems from thediseased Fraxinus sogdianaBunge and elm plants after taining with toluidine blue and DAPI. Theresults show that Aniline blue and DAPI staining can be used to detect Fraxinus sogdianaBungewitches’ broom and Elm witches’ broom.This paper used different methods to test and evaluate six phytoplasmal diseases in Xinjiang, the results can provide technical support and theoretical basis for the prevention of these diseases.