Studies On Detection Methods And Molecular Variation Of The Pathogens Of Sweet Potato Virus Disease (SPVD)

Sweet potato virus disease(SPVD), one of the most important viral disease of sweet potato, is caused by the synergistic interaction between Sweet potato feathery mottle virus (SPFMV) and Sweet potato chlorotic stunt vims (SPCSV).It causes very large reductions in both foliage and root yield of plants, generally reducing yield by above90%,even no yield. Therefore, simple, fast and effective detection of SPVD is essential to the monitoring and early warning. These studies researched the detection methods and molecular variation of the pathogens of SPVD. the main results were summaried as follows:(1) A multiplex reverse transcription polymerase chain reaction (multiplex RT-PCR) method was developed for the simultaneous detection and discrimination of Sweet potato feathery mottle virus (SPFMV) and Sweet potato chlorotic stunt virus (SPCSV), the two pathogens of SPVD. Four compatible sets of primers specific for each virus were designed in conserved regions of the Hsp70gene of SPCSV and the coat protein (CP) gene of SPFMV for use in multiplex RT-PCR assay. The main impact factors of multiplex RT-PCR including annealing temperature, Tag DNA polymerase, dNTPs, Mg2and primers concentration were optimized for the highest sensitivity and specificity. The multiplex RT-PCR method could distinguish the type of the main strains of SPFMV effectively. The sensitivity tests showed that the minimum detectable concentration of SPFMV-CH2,SPFMV-CH and SPCSV were1.42×104copies/μL,1.32×104copies/μL and2.47×104copies/μL respectively. This method could be used for the detection of virus in sweet potato plants, tubers and Bemisia tabaci, which provided a useful tool for the monitoring and early warning of SPVD..(2) The methods of NCM-ELISA, RT-PCR and semi-nested RT-PCR were used to detect Sweet potato chlorotic stunt virus(SPCSV) in whiteflies(Bemisia tabaci) and the sensitivity of the methods above were compared. The results showed that,SPCSV can be detected from at least16whiteflies with virus by NCM-ELISA, from single occasionally and more than three steadily detected by RT-PCR;SPCSV can be steadily detected from single insect by semi-nested RT-PCR. With the transcript RNA in vitro as template, the sensitivity were up to5.69×104copies/μL and5.69×101opies/μL by RT-PCR and semi-nested RT-PCR respectively, which indicated that the detection sensitivity of the semi-nested RT-PCR was1000times higher than that of the RT-PCR. (3) Virus samples were collected from14provinces(cities) in China.The coat protein (CP) gene and RNA13’end gene of Sweet potato chlorotic stunt virus(SPCSV)were amplified, sequenced and analysed. Phylogenetic analysis of CP gene showed that18isolates all belonged to SPCSV West African (WA) strain and shared98.4%-100%identities in nucleotide level,which indicated that CP gene of SPCSV shared a high degree of sequence conservation among different regions of China. Phylogenetic analysis of3’end gene showed that isolates of anhui,jiangshu-2and chongqin shared above99%identities in nucleotide level,but only84.4%identities with zhejiang-2isolate, belonged to WA and EA strain respectively,which indicated that there were at least2strains of SPCSV in China.