Molecular Biological Identification Of Tomato Begomovirus And Spider Lily Orthotospovirus In Fujian Province

In order to find out the virus species infecting tomato and spider lily in Fujian Province,the samples of tomato or spider lily from Fuzhou,Putian,Quanzhou,Xiamen,Zhangzhou,Sanming,Ningde with typical viral disease symptoms were detected by molecular biology.The results are as follows:Geminivirus is a kind of single stranded circular DNA virus with twinning particles,which can cause virus diseases in many important economic crops.It has been found in various regions of China,causing serious damage to tomato,tobacco,papaya and other crops.From 2016 to2019,it was found that tomatoes in many areas of Fujian Province showed typical symptoms of geminivirus,such as curled leaves,swollen veins,etc.We took 5 samples of tomato from Fuzhou,Putian,Quanzhou,Xiamen,Sanming,Ningde,Zhangzhou,respectively,and extracted the total DNA of tomato plants.We used the specific degenerate primer PA /PB of geminivirus for PCR amplification.The results showed that except Ningde and Lianjiang of Fuzhou,all the samples in other areas obtained approximately 500 bp specific fragments.Through the homology comparison of NCBI BLAST,it was found that they had high homology with the isolate of Tomato yellow leaf curl virus(TYLCV),and their homology was more than 98.0%.According to the known fragments,specific primers were designed to amplify the remaining part of TYLCV.One or two full-length sequences were amplified in each region,and about 2.7 kb fragments were obtained.Sequence analysis showed that the TYLCV full-length sequence had typical structure of geminiviruses.The virus strand and complementary strand encoded 6 proteins,which were V1,V2,C1,C2,C3 and C4 respectively.At the same time,no specific bands were detected by using specific primers for the geminivirus betasatellite.Orthotospovirus is a kind of plant multi-component negative RNA virus with wide host range and serious damage,which can infect tobacco,tomato,potato and other food crops.In 2019,we found that spider lilies in Fuzhou area of Fujian Province showed typical ring spot symptoms.We extracted the total RNA of plants and amplified it by RT-PCR using the specific J13 and UHP of orthotospoviruses.The results showed that the specific fragments were amplified from all the susceptible samples and identified as part of the sequence of Hippeastrum chlorotic ringspot virus(HCRV).According to the known fragments,specific primers were designed to amplify the remaining part of HCRV,and finally the full-length sequence of Fuzhou isolate was obtained.Sequence analysis showed that the full-length sequence of HCRV has a typical structure of orthotospoviruses,including S,M and L RNA molecules,inwhich S segment encodes an NSs protein and an N protein,M segment encodes an NSm protein and a precursor protein of Gn and Gc,and L segment encodes an Rd Rp protein.It was found that HCRV could systematically infect Nicotiana benthamiana and cause chlorosis,curl,and blight symptoms.In addition to Fuzhou,plants of spider lily in Putian,Xiamen,Sanming and Zhangzhou were found to be susceptible.The total RNA of the samples was amplified by RT-PCR with the designed specific primers,and the specific fragments were obtained.After cloning and sequencing,sequence analysis showed that spider lilies in these five areas were all infected by HCRV.The results of this study will provide an important theoretical basis for the prevention and control of TYLCV and HCRV.