Detection Of Grapevine Viroids

Viroids are single-stranded covalently closed RNA molecules ranging from 239 to 475 nucleotides, with self-replication capabilities, but not encoding proteins. It is regarded as the smallest pathogen by far. Grapevine is an important economic crop in china and now facts have proved that it can be infected by five kinds of viroids: Grapevine yellow speckle viroid-1(GYSVd-1), Grapevine yellow speckle viroid-2 (GYSVd-2), Australia grapevine viroid (AGVd), Hop stunt viroid (HSVd), and Citrus exocortis viroid (CEVd). Among these viroids, only GYSVd-1 and GYSVd-2 can cause apparent symptom when the grapes are infected while others present latent infections in grapevines. However, CEVd and HSVd can cause diseases in other economic crops and reduced their economy value. Viroids can be widely spreaded by pruning, grafting and other agricultural operations. The most effective control method is to uproot the infected plants, but this will seriously affect their economic benefits as a result. Therefore, the detection of grapevine viroids will be very meaningful. The thesis is for the purpose of providing methods of grapevine viroids molecular detection, establishing and improving detection system of grapevine viroids based on grapevines we collected. We also provided the variations among the viroids in different hosts and regions through sequence analysis of AGVd which is infrequent. Our research can also lay the foundation for future research of viroids'pathogenesis mechanism and its relation of co-evolution with hosts.We perpared three RNA and DNA probes of GYSVd, HSVd and AGVd respectively and the results proved that the sensitivity of RNA probe is 1,000 times than DNA probe. We detected 65 samples collected from Beijing and Xinjiang. First , low molecular weight RNA was extracted from these samples, and then we detected viroids using hybridization .The results showed that: 38 samples were infected with GYSVd including 35 samples from Beijing and 3 samples from Xinjiang, and the incidence rate is 58.46 %; 4 samples were infected with AGVd including 2 samples from Beijing and 2 samples from Xinjiang, and the incidence rate is 6.51 %; 50 samples were infected with HSVd including 43 samples from Beijing and 7 samples from Xinjiang, and the incidence rate is 76.90 %;.Primers of GYSVd, AGVd, HSVd and CEVd were designed according to the sequences which were published on GenBank. We screened specific primers for detecting AGVd and HSVd respectively and also screened the specific primers for classifying GYSVd-1 and GYSVd-2; at the same time, two paired primers were designed to detect CEVd.After cloning and sequencing analysis for Jingchuan-1014 and Zaoyu which were proved to be infected with AGVd by dot-blot, we compared all the clones'sequences to the AGVd sequences published on GenBank. The results showed that: AGVd isolated from Beijing samples have the highest homology to AGVd from Xinjiang samples.The the only difference is that the 15th base of all the clones we obtained were changed.The experimental procedure of detecting viroids using Northern-blot was established in this research. 0.75g samples were sufficient to detect HSVd and GYSVd, while 3g samples were sufficient to detect AGVd.In biological indexing, we only detected HSVd from inoculated indicator plant which was inoculated by AGVd and HSVd at the same time, probably the infectivity of AGVd was inhibited by HSVd in indicator plant.