Molecular Identification Of Three Pathogen Of Phytoplasmas Diseases In Xinjiang

The phytoplasmas associated with Tomato big bud, Lactuca sativa yellow and Convol-vulus arvensis cluster occured in xinjiang were identified at molecular levels. The results asfollows:Tomato plants were collected with leaf chlorotic, smaller and twisted; axillary budproliferation; calyx malformation present horn shape; without fruit from field, which wassuspected to be phytoplasma disease. The purpose of this study was to identify the taxonomicstatus of tomato big bud in Xinjiang by molecular methods. On the basis of nucleotidehomology comparion, phylogenetic tree analysis of the TBB phytoplasma16Sr RNA gene,23S rRNA gene fragment and elongation factor (tuf) gene by PCR with other phytoplasmastrains.About1.2kb fragments were amplified with phytoplasma universal primers for16SrRNA gene from the total DNA, then it was cloned and sequenced. On the basis of homo-logous comparison, phylogenetic trees and subjected to RFLP analysis, the results ofhomologous comparison with other phytoplasma16S rRNA gene fragment have listed on theNCBI showed that this phytoplasma strain shared high homology (99.30%) with the phyto-plasma of Clover rosette that belongs to16SrVI group, even high homology (99.72%) withCucurbit phyllody phytoplasma and Tomato big bud. TBB has the same virtual RFLP mapanalysis with AY390261(16Sr VI-A subgroup). Using phytoplasma universal primers for23S rRNA gene and tuf gene by PCR. DNA fragments about2.8kb and0.8kb in length wereamplified from the total DNA of Tomato showing huge bud symptom. The fragments werecloned, sequenced and subjectted to sequence analysis, The23S rRNA and tuf gene of phyto-plasma isolate Tomato big bud (TBB) shared the highest identities of99.2%and100%,respectively, with Elm yellows group phytoplasma (16Sr V group). In the phylogenetic treesconstructed with23S rRNA and tuf genes, the results showed that it has a near evolutionaffiliation, clustering with the phytoplasma Clover rosette (16Sr VI) group by constructedphylogenetic tree. So TBB phytoplasma belonging to16Sr VI group was deduced to be16SrVI-A subgroup initially.Phytoplasma universal Primer Pairs were adopted for16S rRNA gene amplifieation bynested-PCR.1.2kb fragment was amplified from total DNA of Lactuca sativa sampleLSY_WJ-2with chlorisis mottled leaf yellowing symptom. The taxonomic status of Lactucasativa yellows was identified by molecular methods in Xinjiang. Special primers (fTufu/rTufu)of phytoplasma tuf gene (EF-Tu) were used to amplify from DNA sample. On the basis ofnucleotide sequences homology comparion and phylogenetic tree anaysis of the LSY phyto-plasma16Sr RNA gene and elongation factor (tuf) gene, The results indicated that thisphytoplasma strain belongs to Candidatus Phytoplasma ulmi (16Sr V) group, and it wasdeduced to be16Sr V-B subgroup initially.Convolvulus arvensis plants showing leaf chlorotic, roll and smaller, plants displaycluster symptom obviously, which were collected from experiment station of shiheziuniversity in xinjiang. Phytoplasma universal Primer Pairs were adopted for16S rRNA geneamplifieation by nested-PCR.1.3kb length fragment was amplified from total DNA of Convolvulus arvensis sample with cluster symptom, then the amplified fragment was clonedand sequenced. On the basis of phylogenetic tree analysis and nucleotide homologycomparion with other phytoplasma strains, the results showed that the16SrDNA ofConvolvulus arvensis cluster phytoplasma consists of1229bp, which is clustered togetherwith the members of phytoplasma Peanut witches-broom (16Sr II) group, and shared a highsimilarity (98%) with the phytoplasma EBB-A (JX083377), and it was suggested to16Sr II.