Investigation Of Sweet Potato Viral Disease In Southern China And Establishment Of Molecular Detection Method For Its Causing Viruses

China is the largest sweet potato growing countries.Viral disease,which decreases yield and quality even cultivar productiblity,has become a serious problem in sweet potato production due to its vegetably propatagationin recent years.Planting virus-free seedlings is one of the most effective strategies to improve the yield of sweet potato and prevent the viraldisease.Identifition and detection of infecting viruses are the base for virus-free seedling production,therefore we designed this study in South C hina,a major sweet potato producing areasin C hina.In twelve main sweet potato-growing areas of South China,sweet potato virus disease was surveyed and diseased samples were collected.Themain virus types of sweet potato were identified by small RNA deep sequencing and RT-PCR detectiontechnology,And established a multiplex RT-PCR detection technique for simultaneous de tection of seven sweet potato viruses,which laid the foundation for the research of detoxification sweet potato production.The major results as follows:1.sweet potatovirus diseases was surveyed in 12 sweet potato-growing areas,Guangzhou,Luoding,Meizhou,Leizhou,Huizhou,Shaoguan and ZhanjianginGuangdong Province;Baoting and Tunchang in HainanProvince;Nanning,Hezhou and Liuzhou in Guangxi Province.Sweet potato disease symptoms are usually manifested aschlorotic mottled,veins,shrinkage,purple mottled,mosaic,deformed and so on.The sweet potato virus diseases occurred in all of the above-mentioned areas,and caused losswith the disease rate of 1.6% to 51.7%.The main symptoms of diseased plantsin the fields exhibited chlorotic mottled and purple mottled.2.Using small RNA deep sequencing technology and comparative analysis to identify the viruses in sweet potatosamples,nine viruses were detected in thirteen mixed samplescollected from Guangzhou and Zhanjiang,including 7 RNA viruses:sweet potato chlorotic stunt virus(SPCFV)?sweet potato feathery mottle virus(SPFMV)?sweet potato virus C(SPVC)?sweet potato virus G(SPVG)?sweet potato latent virus(SPLV)?sweet potato virus B1(SPVB1)?sweet potato virus B2(SPVB2)and 2 DNA viruses:sweet potato leaf curl virus(SPLCV)?Sweet potato golden vein virus(SPGVV),respectively to detect and verify viruses in the sequencedsamples byRT-PCR.3.According to the results of small RNA sequencing,and reference to other 8 kinds of sweet potato viruses that were reported,total of 17 viruses were tested by PCR or RT-PCR,13 sweet potato viruses were successfully detected in 209 samples collected from 12 sweet potato-growing areas,in addition to the above nine kinds of sweet potato virus that small RNA deep sequencing technology identified,but also includedSweet potato mild mottle virus(SPMMV),Sweet Potato Mild Speckling Virus(SPMSV),Cucumber mosaic virus(CMV)and Tobacco mosaic virus(TMV).According to detection probability,these viruses in proper order were SPVB2(40.67%)?SPFMV(38.28%)?SPVC(30.14%)?SPVG(26.79%)?SPCFV(19.13%)?SPVB1(14.83%)?SPMSV(12.50%)?SPLV(8.13%)?SPGVV(6.22%)?SPMMV(4.31%)?SPLCV(4.31%)?CMV(2.87%)?TMV(0.96%),the predominant virus of sweet potato infection in southern C hina was SPVB2?SPFMV?SPVG and SPVC.Only 62 in 209 samples had onevirus,accounting for 29.67%,and the other samples were mixed infection,The detection rate of mixed infectionwas 70.33%.These results indicated that viruses mixed infection in sweet potato was serious in South C hina.4.Inordertosecuresweet potato's yield andquality,auamultiplex RT-PCRsystemto simultaneous detection of seven sweet potato virus diseases was established.Based on the detection of sweet potato virus disease by single PCR / RT-PCR,a new multiplex RT-PCR method for the detection of sweet potato virus with high efficiency wasexplored.Degenerateprimerpairsweredesigned according to the respective pathogen isolate sequence data available from SPFMV?SPVC?SPVG and SPLV,which all from Potyvirus,the specificity of degenerate primers was verified by PCR,the degenerate primers were combine with other three degenerate primers: CMV?TMV and SPCFV to establish a multiplex RT-PCR detection system.The mRT-PCR system was optimized in terms of the concentrations of the main ingredients such as annealing temperature and primer concentration.With this system were SPFMV?SPVC?SPVG?SPLV?CMV?TMV and SPCFV in mixed infection samples successfully amplified to obtain four specific fragmentsof1100bp?900bp?600bp?300bp,which were the same as the designed ones in size.ThemRT-PCRsystemisabletodetect seven diseasessimultaneouslywhenitis applied topractical thus greatly increasing detection efficiency and reducing detection cost.So thismRT-PCRdetection systemprovidesareliable,sensitive,rapid methodfordetecting sweet potato virus,embodying the advantages of mRT-PCR.