Study On The Distribution Of Tobacco Virus Disease In Different Ecological Areas In China And The Detection Technology For Tobacco Viruses

Tobacco is one of the important economic crops in China,the quantity and quality of tobacco directly affects the economic benefits of tobacco industry.Tobacco virus disease has become an important factor restricting the development of tobacco industry.However,there is a lack of systematic understanding of the distribution of tobacco virus diseases in different ecological areas.In addition,the rapid detection of some common viral diseases in China lacks intensive study.In this paper,the small RNA deep sequencing technology was used to systematically study the occurrence and distribution of tobacco virus diseases in the ecological area of Yunnan Sichuan Plateau、Shandong ecological area as well as Hubei,Chongqing and Guizhou ecological zone in China.Finally,a LAMP/RT-LAMP detection system was established for some common viral diseases.This study mainly draws the following conclusions and innovations:1.The PVY is the most popular virus in Yunnan Sichuan plateau ecological area.The detection rate is 86.64%,and the epidemic trend is large area.In this area,the prevention and control measures for PVY should be strengthened.In contrast to other regions,CMV and TMV are less dangerous in this area,while LBVV is more serious.Besides,ToNSV,PTV and SYSV are also detected in the area.2.TMV,CMV and TEV are the three main types of viruses that harm Hubei,Chongqing and Guizhou ecological zone.The detection rate is 35.83%,14.34%and12.32%respectively.In addition,the TEV detection rate of 12.32%in this region,according to the usual China tobacco plant diseases and insect pests forecasting network data analysis,The TEV have not widespread occurrence in Chongqing and Hubei,So we predict that TEV has already started to be popular in this region.Besides,MDV,TYLCV,Pe VYV,PepMoV and PVCV are also detected in this area.3.TMV,PVY,CMV is still the main virus that damages the tobacco in shandong ecological area,the detection rate are 34.90,23.43%and 25.57%respectively.TVBMV also has a large distribution in Shandong province with a detection rate of 8.96%.Besides,ReMV,GMMV,ToMV,ToAV and TYLCV are also detected in shandong ecological district.4.In this study,we have detected some viruses that have never been reported in tobacco in different ecosystems in China for the first time,such as ToNSV,ZYMV-HB,PVCV MDV-SCLS,PeVYV,Re MV-Shangdong,GMMV and TYLCV-QDJM.We selected ZYMV,ReMV,MDV and TYLCV as the research objects to verify the molecular biology.First,the coat protein gene sequence of ZYMV-HB and ReMV-Shandong as well as the the genome sequence of MDV-SCLS and TYLCV-QDJM were obtained by gene amplification,cloning and sequencing.Finally,phylogenetic tree was constructed to analyze the phylogenetic relationship between these isolates with other isolates from home and abroad.5.Successfully established a sensitive,rapid and convenient RT-LAMP detection system to detect PVY,CMV and TMV.6.In this study,loop-mediated isothermal amplification（LAMP）was developed and used to detect MDV for the first time.The Mg²⁺and dNTP concentrations and the reaction temperature and time of the LAMP were optimized to 8 mM,1.8 mM,65°C,and 60 min,respectively.The best ratio of the inner primers（FIP and BIP）to the outer primers（F3 and B3）was 2:1.The detection limit of the method was 100 times greater than that of polymerase chain reaction（PCR）.Thus,we established a visual detection system using LAMP.The nucleotide amplification could be clearly observed by adding SYBR Green I,and the positive and negative reactions exhibited distinctly different colors in daylight;however,the positive reactions can present as green fluorescence under a UV lamp.To sum up,small RNA depth sequencing technology relies on its sensitivity,and big data analysis provides a powerful tool for plant virus detection.In this study,we have used this technology to clarify the distribution of tobacco virus diseases in different ecological areas in China.In addition,LAMP/RT-LAMP detection system was established for some common viral diseases.Therefore,these studies provide a theoretical basis for the prevention and control of tobacco virus diseases in China.