Genetic Diversity And Infectious Clone Construction Of Tomato Yellow Leaf Curl Virus In China

Tomato yellow leaf curl virus (TYLCV) is a member of Begomovirus that belongs toGeminiviridae, and It’s transmitted exclusively by the B-type and Q-type whitefly, Bemisia tabaci.Tomato yellow leaf curl disease (TYLCD) is caused by TYLCV, and has made a dramatic damage in allof the subtropical and tropical regions, and it had become an important limit factor in tomato productionof the world tomato industry. In China, TYLCV was first reported in the field cultivation tomato ofShanghai in2006. A few years later, That virus spread rapidly to the provinces of Shandong, Zhejiangand other regions, and had a large scale outbreaking in that areas, It serious harm to the tomatoproduction.In order to clarify the types of TYLCV that infected tomato in China, and establish a rapiddetection system of TYLCV, and try to find silence suppressor of TYLCV at the genetic level, and at thesame time completed TYLCV infectious clone, and provide a theoretical basis for further research indefending and controlling of this diseases. We collected22samples that suspected infection TYLCVfrom more than ten provinces in China in2012, We study it with the pathogenic molecular identificationand sequence alignment analysis.Using the universal primer pair PA/PB of geminivirus for PCR amplification from22samples often provinces. We can amplify a500bp specific fragment. Descriptions, In the main producing areas ofthe ten provinces were infected by geminivirus.The PCR product was purified, cloned and sequenced,sequence alignment, preliminary identification of19samples were infected by TYLCV, and3sampleswere infected by TYLCCNV. We redesigned a specific primers pair of TY-F/TY-R based on thesequence results, and a specific detection of PCR system was established to detect the TYLCV.Based on the accurate sequences, a primer pair was Full-F/Full-R was designed and used toamplify the complete DNA-A sequences of the19samples that was infected by TYLCV. After cloningand sequencing, and phylogenetic analysis showed that the19TYLCV isolates has97.0%to99.7%similarity. And also has a high similarity with other regions of TYLCV isolates from the Genbankdatabase. And them have a typical Begomovirus gene structure, suggesting that their isolates areTYLCV. We using the primer pair DB-F/DB-R to detect the virus DNA-B component, and non DNA-Bcomponent were found from the22samples. At the same time, an universal primer pair Dβ-F/Dβ-R wasdesigned to detect the22samples, and only Guangxi, Sichuan’s three samples were detected of DNAβ,and that three samples also were infected by TYLCCNV. Therefore this satellite DNAβ is probably necessary to TYLCCNV to induce typical disease symptoms in host plants. And It formed of a diseasecomplex of TYLCCNV/DNAβ to harm the host plants.Multiple sequence alignment of the complete found that although TYLCV occurs generallythroughout the worldwide, But all of the TYLCV isolates shared a high similarity, and has a lowermutation rate compared to other begomovirus. We made a phylogenetic analysis with TYLCV isolatesof various regions in China from2008to2012from GenBank database, Indicate that the virus with atypical seasonal epidemic dynamics. We find that TYLCV infected in the eastern provinces andsoutheastern coastal areas, such as HebeiProvince, Henan Province, Anhui Province, ShandongProvince, Jiangsu Province, Zhejiang Province, Shanghai and other regions. And TYLCCNV infected insouthwest China and western areas, such as Guangxi Province, Sichuan Province, Yunnan Province,Xinjiang Province.The rules of diversity of population from different region or in different time wereclear. The genetic distance analysis of TYLCV isolates within and between the provinces showed thatthe diversity of isolates hasn’t distinct geographical characteristics and has formed a significant straindifferentiation, the virus showed aggregative distribution and It’s outbreaks were caused by manydifferent TYLCV strains by different transmission in China.In order to further study pathogenicity of TYLCV, an infectious clone of pBI121–SD–1.78A wasconstructed by the gene cloning techniques. The results of Agrobacterium-inoculated tobacco, tomatoesand peppers indicated that the inoculated tomato plants with better pathogenicity, and showed yellowleaf curl symptoms. And also showed a certain degree of pathogenicity on Benthamiana, but hasnon-pathogenicity on peppers. The above results suggested that the pBI121–SD–1.78A had infectivity.Using the infectious clone to Agrobacterium-inoculated the TYLCV-resistant materials, We found thatthe three resistant materials exhibits good resistance. By Agroinfiltration identified that the silencesuppressor V2of TYLCV, The V2gene suppress the TYLCV resistance of host plants. The study laidthe foundation for the virus gene function, pathogenicity, infection circle and the control technology.