Identification Of Species, Molecular Variation And Detection Methods Of Sweepoviruses In China

Sweet potato is a important food crops, food processing and industrial raw materials and would be a energy crops potentially in China. China is the largest producer of sweet potato in the world. Diseases infected by sweepoviruses were increasing in recent years and seriously affected the healthy development of sweet potato industry in China. For lack of systematic researches of sweepoviruses in China, the species, distribution, molecular variation and pathogenic of sweepoviruses were unclear. These led to having no scientific basis for prevention and control of this disease. This study focused on resolving these problems and the main results were as followes:1) Species, occurrence frequencies and distribution of sweepoviruses in China:About330sweet potato samples collected from more than20provinces (cities) in China were detected using PCR or RCA methods. A total of51complete or nearly complete genomic sequences of sweepoviruses were characterized. Sweepoviruses in China can be divided into nine species, including three species:Sweet potato leaf curl virus (SPLCV), Sweet potato leaf curl China virus (SPLCCNV) and Sweet potato leaf curl Geogia virus (SPLCGoV); two tentative species:Sweet potato leaf curl Shanghai virus (SPLCShV) and Sweet potato leaf curl Japan virus (SPLCJV); and four new species:Sweet potato leaf curl China Sichuan virus (SPLCCSV), Sweet potato leaf curl Henan virus (SPLCHnV), Sweet potato leaf curl Guangxi virus (SPLCGxV) and Sweet potato leaf curl Henan4virus (SPLCHn4V). SPLCV had the highest frequency37.3%, followed by SPLCShV (17.6%). The frequency of SPLCHn4V and SPLCCNV were11.8%and2.0%respectively and were all relatively low. Sweepoviruses had distributed in three major sweet potato zonings in our country.54.9%of the positive samples showed leaf symptoms.2) Molecular variation of sweepoviruses in China:Nucleotide sequence similarities among51different isolates of sweepoviruses in China were between78.6%-99.9%. Nucleotide sequence variation among19isolates of SPLCV was the largest and the similarities were between91.3%-99.9%. Nucleic acid sequence variations among7isolates of SPLCJV were relatively small and the similarities were between98.7%and99.8%. The evolutionary tree showed that sweepoviruses in China could be divided into nine branches. The64CP genes of sweepoviruses in China had84.5-100%nucleotide sequence similarities and could be divided into two large branches in the evolutionary tree.3) Complete sequences and recombination analysis of four new species:Complete sequences of four new species were obtained. SPLCCSV consisted of2764nts and shared the highest level of nt sequence identity (84.8%) with SPLCCNV; Recombination analysis suggested that SPLCCSV had sequences derived from recombination between SPLCV-GZ01and SPLCV-Merremia N4. SPLCHnV consisted of2766nts and shared the highest nucleotide sequence identity (89.0%) with SPLCCSV; The AC2and AC3regions of SPLCHnV were generated by recombination between SPLCCNV-[CN:05] and SPLCV-CE[BR:Forl], Non-coding intergenic region (IR) had sequences derived from recombination between SPLCBeV and SPLCCNV-ZJ. SPLCGxV consisted of2831nts and shared the highest nucleotide sequence identity (88.3%) with SPLCCSV; SPLCGxV contained sequences derived through recombination with SPLCHn4V-[Sichuan14:2012] and SPLCShV. SPLCHn4V consisted of2826nts and shared the highest nucleotide sequence identity (89.7%) with SPLCCNV; It contained sequences derived through recombination with SPLCV-J508, SPLCV-[Guangxi2:2012] and SPLCV.4) Detection methods of sweepoviruses:①Prokaryotic expressions and antiserum preparations of SPLCCSV-CP and SPLCV-CP:The CP genes of SPLCCSV and SPLCV were efficiently expressed in Escherichia coli respectively. The express proteins were used as antigens to immune rabbits and two specific antiserums were raised. The results of ACP-ELISA showed that the antiserum titer of SPLCCSV was1:5000and that of SPLCV was1:2000. Two antiserums all could be used in detection of sweet potato samples in the field.②Construction of a multiplex PCR detection method:A multiplex PCR method was developed for the simultaneous detection three sweepoviruses:SPLCV, SPLCGoV and SPLCHn4V. Specific primers were designed for these sweepoviruses and the main impact factors of multiplex PCR were optimized. The multiplex PCR method could distinguish the three species of sweepoviruses effectively. The minimum detectable concentrations were2.46×104copies/μL,1.47×104copies/μL and1.58×104copies/μL respectively.③Detection of sweepoviruses based on deep sequencing technology:Deep sequencing technology was first applied in detection of sweepoviruses from sweet potato. Through extracting total DNA of two sweet potato samples, RCA, build DNA library, high-throughput sequencing, data quality control, sequence splicing and PCR verification, seven species of sweepoviruses were detected.5) Construction of infectious clone SPLCCSV:Infectious clone SPLCCSV was constructed using the seamless connection technology. Three weeks after inoculated with agrobacterium, the systematic leaves of Nicotiana benthamiana appeared slight puckering, foaming and yellowing symptoms, SPLCCSV could be detected by PCR methods and the infect activity of SPLCCSV clone had been evidenced preliminary.