| Yam（Dioscorea oppositifolia L.）mainly propagates asexually through tubers,which leads to the accumulation of various pathogens,reduces the quality and yield of yam,and causes economic losses.Yam virus disease is an important disease threatening yam.It occurs in yam growing areas of all countries in the world,and the viruses detected on yam in different countries and regions vary greatly.With the development of detection technology,new viruses are constantly reported.Moreover,the phenomenon of virus mixed infection was widespread in yam.Therefore,it is of great significance to systematically identify the species of yam virus and establish main viruse detection technology in the prevention and control of yam virus disease.Henan is one of the important growing areas of yam in China.In this study,transcriptome sequencing and molecular biological detection techniques were used to detect and identify the virus species of yam in Henan province.The occurrence,main virus species,mixed infection and genetic variation of yam virus disease in Henan province were studied,and the genome sequence of new recorded species was cloned.On this basis,the rapid detection technology of main viruses was established,and the main research results were as follows:1.The main virus species and virus symptoms of yam in Henan province were determined:from 2020 to 2022,188 leaf samples suspected to be infected with the virus were collected from the main production area of yam in Henan province.Using transcriptome sequencing combined with polymerase chain reaction（PCR）techniques,five Yam viruses were identified:Yam yellow spot mosaic virus（YYSMV）,Yam latent virus（YLV）,Broad bean wilt virus-2（BBWV-2）,Japanese Yam mosaic virus（JYMV）and Youcai mosaic virus（YoMV）,the detection rate of JYMV,YoMV,YYSMV,BBWV-2and YLV was 94.15%,87.23%,68.09%,42.02%and 29.79%respectively.The mixed infection rate of two or more viruses was as high as 98.94%,JYMV+YYSMV+YoMV was the main type of virus compound infection.Five isolates of BBWV-2,YLV,YYSMV,JYMV and YoMV were obtained from yam,the nucleotide identities of the isolates were98.36%,96.70%,98.02%,99.02%and 99.48%,the molecular variations of the five viruses were small.The results of field investigation on yam showed that most of the symptoms of yam virus in the field were chlorosis of mosaic,some appeared necrotic spot of mosaicand deformity of mosaic,and there appeared hidden symptom under high temperature conditions.2.In this study,three contigs shared 96.29%-96.72%homology with YoMV-SX（Gen Bank accession number:JX422022）,respectively,by transcriptome sequencing and Blastn analysis.Reverse transcription-polymerase chain reaction（RT-PCR）was performed with four pairs of specific primers to amplify the polymerase chain reaction of YoMV-Do41.Sequence structure analysis showed that the YoMV-Do41 genome was6274 nt in length,encoding four ORFs.The results showed that the identity of YoMV-Do41 with YoMV-SX of Rehmannia glutinosa in Shanxi province was the highest（97.51%）,and that with YoMV-As1-2 of Brassica rapa was the lowest（89.09%）.The phylogenetic tree analysis indicated that YoMV-Do41 was closely related to YoMV-SX.This study is the first report on YoMV infected yam.3.In this study,Reverse transcription loop-mediated isothermal amplification（RT-LAMP）of YLV,BBWV-2 and YoMV was established,RT-LAMP primers were designed according to the coat protein（CP）gene sequences of YLV,BBWV-2 and YoMV.The optimal reaction temperature,specificity and sensitivity were studied,The reaction temperature of YLV and BBWV-2 are 65℃,and that of YoMV is 60℃.The RT-LAMP method of YLV,BBWV-2 and YoMV could detect 5.08×101 copies·μL-1,6.47×103copies·μL-1and 9.18×102copies·μL-1.The specificity of RT-LAMP was very good,it can quickly detect the target virus.4.Virus mixed infection is common in yam.In Henan province,the main viruses on yam are JYMV,YYSMV,YLV,BBWV-2 and YoMV.The specific primers of multiplex PCR were designed according to the conserved sequences of five viruses,a multiplex PCR method for the detection of five viruses was established.The reaction system was 30μL,containing Monamp 1×Taq Mix Pro（+Dye）,DNA or cDNA of each virus were 1μL,the amounts of specific primer pairs for each virus were 0.24μM of YYSMV F/R,0.32μM of YLV F/R,0.16μM of BBWV-2 F/R,0.04μM of JYMV F/R,0.08μM of YoMV F/R,finally,add dd H2O to 30μL.The annealing temperature of the multiplex PCR program was 55.0℃,and the sensitivities of YYSMV,YLV,BBWV-2,JYMV and YoMV were 3.36×105 copies·μL-1,5.08×105 copies·μL-1,6.47×105 copies·μL-1,6.89×105copies·μL-1 and 9.18×105 copies·μL-1.It can quickly and efficiently detect five viruses on yam,and provide technical support for virus disease control and monitoring in yam production. |
