Research On Molecular Characteristic Of Citrus Huanglongbing Pathogen And Viruses Mix Infected, And Elimination By Cryopreservation Protocol

Citrus huanglongbing globally has been regarded as one of the most destructive disease to citrus production. Pothogens easily mix infect with it are Citrus tristeza virus(CTV), Citrus exocortis viroid (CEVd) and Citrus tatter leaf virus (CTLV). Biological and molecular characteristic of these four pathogens were studied. The objective is to establish highly sensitive molecular detection method for their rapid detection; and to study part of their molecular character; another objective is to make sure the possibility and the mechanism of eliminating the above 3 pathogen (HLB, CTV, CEVd) by cryopreservation protocol. The main results are described as follows:1. Biological identification and optimization of PCR system for the identification of HLB pathogen. Several HLB samples were inoculated to Catharanthus roseas and Citrus reticulata Blanco by mechanical method and grafting-inoculation respectively. These isolates caused typical symptoms on indicator plants. PCR, Semi-Nested PCR and Nested-PCR were established by adjusting the main factors in PCR system. Results showed the sensibility of Semi-Nested PCR and Nested-PCR was 10⁴ times higher than single PCR. HIB pathogen (Candidatus Liberobacter asiaticus) was firstly detected out on wampee by Nested-PCR. It is the first report that HLB pathogen can infect wampee throughout the world.2. Comparison of sensitivity of different primer sets for the detection of HLB pathogen. Based on the sequence of rplA/rplK, β-operon, 16SrDNA and 16S/23S rDNA, 5 different primer sets were designed. The detection results showed sensitivity of different primer sets were not identical, from the highest to the lowest were fP400/r P400> fP535/r P535>fA2/rJ5> fOIl/rOI2c> fOI2/r23S1.3. Identification of Candidatus Liberobacter asiaticus, amplification of 16SrDNA, the consequent analysis of its products by RFLP, SSCP and phylogenetic tree analysis for 16SrDNA. Results showed that 9 different representative isolates coming from 7 provinces were all belong to Candidatus Liberobacter asiaticus. Sequence analysis of 16S/23SrDNA intergenic region and phylogenetic tree constructed showed that 18 representative isolates coming from different hosts, showing different symptoms (7 provinces) had no variation; the homology among isolates was above 99%. They were all highly homologous with Candidatus Liberobacter asiaticus, and distinct from Candidatus Liberobacter africanum.4. Detection of HLB pathogen by Southern blotting. The α-³²P-dCTP labeled probe of...