Disease Survey,Diagnosis,and Molecular Characterization Of Viruses Infecting Yam In China

Viral diseases are one of the most important factors limiting the production of yam in China by causing heavy loss in yield and decline in quality.A three-year survey conducted in the major yam growing provinces of Guangxi,Jiangxi,Henan,Shandong and Jiangsu provinces in China revealed that viral diseases were widely distributed and an incidence of 7%-40%was observed on varieties belonging to D.alata and D.japonica in Guangxi and Jiangxi of the southern China,and 2%-59.3%on varieties belonging to D.opposita cultivated in Henan,Shandong and Jiangsu in the North of China.Viruses of Potyvirus,Badnavirus,Potexvirus and Cucumovirus genera have been reported to infect yam world-wide.In this study,the causative viral agents were identified by small RNA deep-sequencing and PCR/RT-PCR of 365 leaf samples collected from the 5 provinces mentioned above.Dioscorea bacillifrom virus(DBV)of genus Badnavirus,Yam mild mosaic virus(YMMV)and Japanese yam mosaic virus(JYMV)of genus Potyvirus,Yam virus X(YVX)of genus Potexvirus,Yam latent virus(YLV)of genus Calarvirus,Chinese yam necrotic mosaic virus(CYNMV)and Yam chlorotic necrotic mosaic virus(YCNMV)of genus Macluravirus,Broad bean wilt virus 2(BBWV-2)of genus Fabavirus,and Yam yellow spot mosaic virus(YYSMV)of genus Begomovirus were detected.DBV,YVX,YMMV,JYMV,CYNMV and YCNMV were found in D.alata;DBV,YYSMV,YMMV and JYMV were found in D.japonica;and DBV,YYSMV,YVX,YMMV,JYMV,BBWV-2,CYNMV and YLV were found in D.opposita Thunb.Further,it was noticed that DBV(71.1%),YMMV(49.3%)and YVX(16.2%)were frequently detected on D.alata;DBV(66.7%)and JYMV(12.6%)were frequently detected on D.japonica;JYMV(43.4%),YLV(31.6%),DBV(19.9%),YMMV(17.6%),BBWV-2(17.6%)and YYSMV(12.5%)in D.opposita.The positive detectable rate of other viruses in the these three yam species were less than 5%.Double or triple infections were detected in 138 of 365 samples which indicated that co-infection or multi-infection prevalent in yams.A sample from D.opposita cv.SSY was found to be infected by 5 viruses,simultaneously.The complete genomes of isolates YMMV-NC1,JYMV-WX1,CYNMV-FX1,BBWV-2-SG,YVX-WX1,YLV-SG1 and YYSMV-FX1,were determined by using RNA-Seq,RACE and RT-PCR.The genome organizations of the novel viruses YVX-WX1,YLV-SG1 and YYSMV-FX1,were found to be the same to that of Potexvirus,Carlavirus and Begomovirus,respctively.YVX-WX1 shared the highest similarity to White clover mosaic virus(WCIMV)Japanese isolate with 53.1%nucleotide identity.YLV-SG1 shared the highest similarity to Hop mosaic virus(HpMV)Australian isolate with 62.9%identity at nucleotide level.And YYSMV-FX1 shared the highest similarity to Passionfruit severe leaf distortion virus(PSLDV)Brazil isolate with 54.2%nucleotide identity.A phylogeny tree with 26 YMMV isolates collected in this study and 19 other YMMV isolates deposited in the GenBank was constructed.These isolates fell into 5 groups with geographic origin corelation.Isolates detected in the South and the North of China belonged to two separate groups,which were far from each other in the phylogenetic tree,suggesting that YMMV evolution may be affected by geographic isolation,yam cultivars and recombination events.Recombination hotspots were detected in the P1 to C1 region of YMMV.Monoclonal antibody against JYMV was successfully generated by SEAL,and CP polyclonal antibodies against the YMMV,YVX,CYNMV and YLV were generated using the bacteriam-expressed viral coat proteins.Except for the JYMV monoclonal antibody that exhibited a slightly lower specificity with a light background,the specificities of the polyclonal antibodies against other viruses were high.The titers of all antibodies generated were higher than 1:16000,and when the infected leaf sap was diluted 1000 folds,a strong immune reaction could still be observed with the viral polyclonal antibody.ELISA and IC-RT-PCR protocals were developed,viruses could be detected in the infected leaf sap diluted up to 10-3 by ELISA,and diluted up to 10-6 times by IC-RT-PCR.Multiplex PCR systems for diagnosis of YMMV,JYMV,CYNMV and YLV were established by using adapter primers.In conclusion,incidence of viral diseases on yam and the identities of the causative viruses in the five major yam producing provinces in China were determined this study.At the meantime,sensitive protocals for detecting yam-infecting viruses were developed.Combined together,these developments laid a solid foundation for yam production in the future.