| This dissertation comprises three major parts: paulownia witches'-broom phytoplasma (EF-TU) tuf gene analysis, investigation of alternative host plants of the paulownia witches'-broom phytoplasma as well as the molecular detection of other phytoplasmal diseases from two plants.Two specific primers for phytoplasmal tuf gene were designed according to sequences available in the GenBank. The 1185 bp-tuf gene of PaWB-NY, which encodes the EF-Tu of 394 amino acids (43kDa), was amplified by PCR. The results of TMHMM2.0 and PROSTâ…¡analysis presume that EF-TU is a cytoplasmic protein and is not transmembrane one. The tuf gene of PaWB-NY was ligated to expression vector pGEX-4T-3 and transferred into E. coli BL21 (DE3). SDS-PAGE showed that PaWB-NY tuf gene was expressed as a 62 kDa fusion protein when induced with IPTG. Specific antiserum against PaWB EF-Tu was produced after immunizing rabbit five times with the recombinant protein. The results of ACP-ELISA and dot blotting test using different antigen suggested that the EF-TU might be combined with the membrane, treating antigen with freeze and thaw is essential. Western blotting and indirect immunofluorescence analysis results indicated that a disease-specific polypeptide was detected in PaWB, PeV and CWB-infected plants, but not in healthy plants or JWB, CWB phytoplasma-infected plants.Investigation and identification of alternative host plants of the paulownia witches'-broom phytoplasma were conducted by sensitive molecular detection method. 30 samples, which were collected from Shandong, Henan and Beijing in different time, were detected with Nested-PCR. 1246bp-16S rRNA gene product of phytoplasma was amplified from 8 species plants with or without symptoms. Sequenced phytoplasmal 16S rRNA gene in these plants showed the highest similarity with 16S rDNA of PaWB phytoplasma, sorted to 16SrI-D group. It is the first time to report that Eleusine indica, Setaria viridis, Dioscorea opppsita, Physalis angulata, Cucurbita moschata, Catalpa bungei, Capsicum annuum and Arachis hypogaea could be infected by PaWB phytoplasma. The phytoplasma of CaWL and AhY were obviously different from reported pepper stolbur disease and peanut witches'-broom phytoplasmas respectively. The phytoplasmal tuf gene was also amplified from Catalpa bungei yellow (CbY) leaf, and the result of sequence analysis showed that it shared high similarity with PaWB-BJ phytoplasma which was nearby with it. Indirect immunofluorescence- microscopy results indicated that a disease-specific polypeptide of PaWB-NY tuf was detected in the CbY leaf vein phloem tisssues. The results of DAPI fluorescence microscopy, indirect immunofluorescence microscopy as well as PCR detection demonstrated that the phytoplasma concentration of CbY plant was generally far lower than PaWB infected paulownia.Syringa oblate Yellow (SoY) plant, Syringa oblate proliferation (SoP) plant and Sopnorajaponlca cuchlnensis internode shorten (ScIS) plant were collected from Chengde Hebei province and amplified 1246 bp-16S rRNA gene of phyotoplasmal with nested-PCR. Then, we confirmed the classified position of SoY, SoP and ScIS phytoplasmas by sequenceing and Computer-simulated restriction fragment length polymorphism (RFLP) analysis. This is the first time to report Sopnorajaponlca cuchlnensis infected by the phytoplasma which belongs to 16SrI-D group. Homologous and Computer-simulated RFLP analysis indicated that SoY phytoplasma was like crotalaria witches'-broom phytoplasma, belonging to 16SrII-A group, which was different from lilac witches'-broom phytoplasma. PCR and RFLP analysis of 16S rDNA revealed that the SoP plants were infected by two mutually distinct phytoplasmas. One was share high similarity with paulownia witches'-broom phytoplasma, belonging to 16SrI-D group. Another one belonged to 16SrII-A group. It is the first time to report that Syringa oblate can be infected by mixed phytoplasmas which belong to 16SrI-D group and 16SrII-A group respectively.
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