Molecular Detection And Identification Of Several Different Phytoplasmas From Woody Plants In China

Molecular Detection and Identification of Different Phytoplasmas from Woody Plants in ChinaLi yong Directed by Tian GuozhongThis paper comprises three major parts: the molecular detection of phytoplasma diseases, the compared identification of different phytoplasmas from several woody plants in China and the Extrachromosomal DNA analysis of paulownia witches'-broom.Jujube witches'broom phytoplasma (JWB) was surveyed from natural wild Jujube(Zizyphus spinosa Hu) for the first time. Phytoplasmas were detected by PCR with universal primes from natural wild jujube, cultivated jujube, several jujube species with resistances against JWB, jujube and paulownia tissue culture plantlets after treatment of pathogen elimination. The detection results showed that JWB is very prevalent in natural wild jujube and cultivated jujube. 10-35% of natural wild jujube and cultivated jujube without symptom were shown haurboring the pathogen of JWB in the JWB occurring area. Cultivar Huping jujube and Hama jujube are proved to be high resistance to JWB in the several Jujube species from Hebei province. Between the two batch of the jujube tissue culture material supposed got rid of pathogen from Zhengzhou city were detected for JWB pathogen, there are JWB pathogen in all the four samples from the first batch, but no JWB pathogen was detected in all the four samples from the second batch. Moreover, we have developed PCR-detection Kit â…  and â…¡ to make the dectection easy and fast.Chinaberry witches'broom(CWB), Ziziphus jujube witches'broom(WJWB), Mulberry dwarf disease (MDD), JWB and Paulownia witches' broom(PaWB) phytoplasmas were identified by comparative studies of PCR amplify of phytoplasmal 16SrDNA,23rDNA and ribosomal protein(rp) gene, heteroduplex mobility assay (HMA), RFLP of PCR-amplified products (16S rDNA), cloning and sequence analysis of 16SrDNA and rp gene. An efficient procedure for rapid identification and differentiation of phytoplasmas was established, which could identify and differentiate phytoplasma collected from field. Based on the results of 16SrDNA and rp gene sequence analysis, CWB and SWB in China were identified and sorted as Aster yellows group (16SrI) and Elm yellows group(16SrV) respectively for the first time, and JWB and WJWB were proved to be the same pathogen.The PaWB phytoplasma cells were extracted and isolated using in vitro cultured plantlet with phytoplasma as propagating material. According to the extraction and purification of phytoplasmal DNA, agrose electrophoresis and moleculer hybridization,six extrachromosomal DNA segments were detected from paulownia wicthes'broom samples, their molecular weight are 8kb, 6kb, 3.5kb, 2.4kb, 2.2kb, 1.8kb.The cloning, sequencing and function analysis of the extrachromosomal DNAs need to be further studied.