Transmission Characteristics Of Cucurbit Chlorotic Yellows Virus By Bemisia Tabaci

Sweet potato whitefly Bemisia tabaci(Gennadius)(Hemiptera:Aleyrodidae)is one of the most destructive phloem-feeding insect pests worldwide,biotype B(Middle East-Minor Asia l genetic group)and biotype Q(Mediterranean genetic group)are two of the most invasive and widely distributed whiteflies which transmitted hundreds of plant viruses around the world,causing severe damage to crops by direct feeding and by transmitting 208 plant viruses.Cucurbit chlorotic yellows virus(CCYV),as a recently reported crinivirus,is transmitted by B and Q biotypes of B.tabaci in a semipersistent manner.Currently,studies on CCYV were mainly on the detection methods and resistance breeding,but the mechanism of transmission is still unclear.In this study,with Q biotype B.tabaci and CCYV as an experimental model system,a sensitive real-time RT-PCR method was established to test virus in individual whitefly and in the plant.Characteristics of whitefly-transmitted CCYV was studied,in order to provide foundation for systemic elucidation of the mechanisms of virus transmission by whiteflies and thereby for control of plant virus.The main results of this study are as follows.(1)To detect the amount of CCYV in individual B.tabaci,a sensitive real-time RT-PCR using the green fluorescence dye SYBR Green I was established in this study.Serial 10-fold dilutions of plasmid containing target fragment were used as templates for real-time RT-PCR to construct a standard curve,and the sensitivity and repeatability of this assay were analyzed.This assay was applied to detect the amount of CCYV in individual B.tabaci fed on CCYV-infected cucumber plants for 3 days.The results showed that the sensitivity of real-time RT-PCR was 100 times higher than that of conventional RT-PCR.Sixty amounts of CCYV in individual B.tabaci ranged from 1.34×104to 1.09×107 copies,and the number of copies were mostly concentrated in 104-106 copies.(2)To elucidate the mechanism of transmission,acquisition access period(AAP),retention time,efficiency of transmission and inoculation access period(IAP)of B.tabaci biotype Q were determined by real-time RT-PCR which we had estabished.The results indicated that the minimum AAP was 1 h and the proportion of viruliferous whiteflies reached 100%at 48 h of AAP.In our study,CCYV can be retained in vectors as long as 12 d,and the efficiency of vector transmission was low.The virus was nontransmissible with 30 couples of whitefly for 1 h of IAP and after 6 h of IAP the vector reached low level of transmission efficiency,while after 24 h of IAP the vector reached high level of transmission efficiency.The results confirm that CCYV is transmitted by B.tabaci in a semipersistent manner.