Identification Of Sweetpotato Viruses In Hainan Province And Development Of RPA-LFD Method For Rapid Virus Detection

The development of sweet potato industry has unique advantages in Hainan of China due to its superior climatic conditions.However,virus disease is a major constraint causing significant sweet potato yield losses and poor quality in Hainan.In order to understand the prevalence and occurrence of sweetpotato viruses in Hainan,253 symptomatic samples we re collected from 16 locations in Hainan to identify the viruses infecting sweet potato by s mall RNA deep sequencing and PCR detection.The main typical symptoms,distribution,p hylogenetic relationships and coinfection of sweet potato viruses in Hainan were characteri zed.These results will help to develop management strategies to control spread of sweet po tato viruses in Hainan.Furthermore,rapid detections of sweepoviruses and sweet potato po tyviruses by recombinase polymerase amplification combined with lateral flow dipsticks（RPA-LFD）and reverse transcription RPA-LFD（RT-RPA-LFD）were established,which pr ovide two rapid,sensitive and specific diagnostic tools that are applicable in field and reso urce-limited settings.The main findings are as follows:1.Identification of sweet potato viruses in HainanA total of 253 sweet potato symptomatic samples were collected from 16 locations in Hainan of China and 9 typical symptoms（vein-cleaning,leaf curl,plant dwarfing,mosaic leaves,chlorosis,golden veins,purple pattern,crinkle,and sector）were found.To identify viruses that are infecting sweet potato in fields of Hainan,16 mixed samples were used for the small RNA deep sequencing and PCR/RT-PCR and six RNA viruses sweet potato feathery mottle virus（SPFMV）,sweet potato virus C（SPVC）,sweet potato virus G（SPVG）,sweet potato virus 2（SPV2）,Sweet potato chlorotic fleck virus（SPCFV）,cucumber mosaic virus（CMV）,and four DNA viruses,sweet potato leaf curl virus（SPLCV）,sweet potato golden vein associated virus（SPGVa V）,sweet potato badnavirus A（SPBVA）,sweet potato badnavirus B（SPBVB）were detected.These viruses belong to 5 virus genera and 5 virus families.SPFMV had the highest incidence with71.5%of tested samples.Phylogenetically,three strains of SPFMV EA,RC and O were detected in 13 different locations in Hainan except Baisha,Qiongzhong and Wuzhishan.SPLCV were detected in 64%of the 253 samples in 12 different locations in Hainan.SPVC can be tested in 5 places of Haikou,Chengmai,Wenchang,Wanning and Dongfang;SPVG,SPV2,SPCFV,CMV,SPBVA,SPBVB were only found in one area,respectively.it was found that coinfection with sweet potato viruses was common in Hainan and most samples were simultaneously infected by two to six different viruses.The occurrence of virus disease and virus species in seven main sweet potato varieties（Xinxiang,Guangshu87,Chuanshanzi,Ningzishu,Shulu No.1,Gaoxian 14 and Sanjiaoning）were different.These results indicate that viruses infecting sweet potato in Hainan are diverse.2.Development of rapid and visual detection of Sweepovirusess and sweet potato potyviruses by recombinase polymerase amplification combined with lateral flow dipsticks（RPA-LFD）To develop an RPA-LFD for Sweepovirusess detection,the specific RPA primers and probe were designed based on a conserved fragment of the sweepovirus coat protein gene,and the optimal reaction conditions were 37°C for 20 min.The RPA-LFD specifically detected different sweepovirus species,and there were no cross-reactions with other viruses infecting sweet potato.The RPA-LFD detection limit was 1.0×10⁴copies of the target DNA molecule per reaction,and its sensitivity was 10 times that of conventional PCR.Moreover,a reverse transcription RPA-LFD（RT-RPA-LFD）was established for rapid detection of four sweet potato potyviruses（SPFMV,SPV2,SPVC and SPVG）.The assay.The minimal detection limit of the RT-RPA-LFD assay was 1.0×10^-8μg total RNA per reaction within 20 min at 40°C.The assay was SPFMV,SPV2,SPVC and SPVG-specific since it had no non-specific reactions with other sweet potato viruses.Furthermore,combined with an alkaline polyethylene glycol-based DNA/RNA extraction method,the entire process of detection of Sweepovirusess and sweet potato potyviruses could be completed within 30 min without specialized equipment.Both RPA-LFD methods were successfully applied to detect the Sweepovirusess and sweet potato potyviruses in the field and will be might be promising tools for quick diagnosis of virus-infected sweet potato plants.