| As a characteristic economic crops in Northeast China,shallot（Allium cepa var.aggregatum）has good health function and is favored by consumers.Shallot mainly adopts asexual reproduction in the production,which is susceptible to a variety of viruses,resulting in genetic depression and serious yield reduction.There are currently no effective agents to control shallot virus disease,so it is an important prerequisite to clarify virus infection in different planting areas of shallot and establish an efficient virus detection technology.Therefore,in this study,the duplex RT-PCR method was used to investigate four viral infections of Shallot yellow stripe virus（SYSV）and Shallot virus X（SVX）,Onion yellow dwarf virus（OYDV）and Shallot latent virus（SLV）in Jilin and Heilongjiang shallot planting areas.Real-time fluorescence quantitative PCR methods were established for OYDV and SLV with high detection rates,and the accumulation amount of two viruses in shallot after single infection and mixed infection at different inoculation time was determined by the above methods.It can provide a reference for the prevention and control of shallot virus disease and intensive research of viral pathogenesis.The main results are as follows:（1）Specific primers were designed according to genomic sequences of four viruses,255 samples from Jilin and Heilongjiang shallot planting areas were detected by duplex RT-PCR.229 out of 255 samples were positive,and the incidence of virus disease was 89.8%,of which 87.06%were infected with SLV,36.86%with OYDV and 0.78%with SYSV.Coinfection of SLV and OYDV was the main type of multiple infections with the infection rate of 33.3%,and the mixed-infection of SYSV,OYDV and SLV was the main type of infection containing three kinds of viruses with an incidence of 0.78%.However,no four viral coinfection was detected.（2）A method of real-time fluorescence quantitative PCR was established by using a pair of specific primers according to conserved sequence from the coat protein gene of OYDV.The standard curve Ct value and logarithmic of template copy number showed a good linear relationship.The amplification efficiency was 95.921%and correlation coefficient was 0.996.There was no crossing reaction with Shallot virus X（SVX）and Shallot latent virus（SLV）.The lowest detection limit was 2.0×10~2copies·μL-1,about 1000 times higher than that of routine RT-PCR.Both variable coefficients of intra-assay and inter-assay were less than 2%.This experiment utilized this method and detected 45 samples collected respectively from suspected infectious shallot and garlic.The positive rates were 40.00 and 6.67 percentage point higher than those by conventional RT-PCR assay,respectively.（3）A method of real-time fluorescence quantitative PCR was established by using a pair of specific primers according to conserved sequence from the coat protein gene of SLV.The standard curve Ct value and logarithmic of template concentration showed a good linear relationship.The amplification efficiency was 94.529%and correlation coefficient was 0.9996.There was no crossing reaction with Shallot yellow stripe virus（SYSV）,Onion yellow dwarf virus（OYDV）and Shallot virus X（SVX）.The lowest detection limit was 10-7dilution fold,about 1000 times higher than that of conventional RT-PCR.The variable coefficients of intra-assay and inter-assay were0.02%～0.63%and 0.03%～1.24%respectively.This experiment utilized this method and determine the content of SLV in shallot leaves and bulbs and to detect 50 samples suspected of SLV infection.The content of SLV in leaves was significantly higher than that in bulbs,and 46 out of 50 samples were detected to be positive by this assay.The positive rate was 6.00 percentage point higher than that of conventional RT-PCR.（4）The aggravation of shallot virus disease symptoms and increase of accumulation of two viruses were caused by co-infection of OYDV and SLV.The symptom observation indicated that shallot co-infected by two viruses showed more serious dwarfing and yellowing.In addition,the fresh weight of bulbs co-infected by two viruses was 83.9%lower than that of healthy shallot,9.5%lower than that of OYDV-infected shallot,and 53.6%lower than that of SLV-infected shallot.In this study,we found that the accumulation of OYDV and SLV in co-infected shallot with two viruses was higher than that in single-infected shallot.Therefore,it is preliminarily speculated that OYDV and SLV may have a synergistic effect in co-infected shallot. |
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