Study On Real-Time RT-PCR Technology For Citrus Tatter Leaf Virus (CTLV) Detection And Influence Of Citrus Cultivars On The Composition Of CTLV

Citrus tatter leaf disease caused by Citrus tatter leaf virus(CTLV) is an economically important systemic disease of citrus in the world,and it was first discovered in 1962 in latently infected Meyer lemon trees.CTLV is the species of the genus Capillovirus.The genome of CTLV consist of plus-sense,single-stranded RNA.CTLV is transmitted by graft and mechanical inoculation.CTLV-infected cultivars on trifoliate orange or its hybrids are usually stunted and chlorotic,and they also show a pronounced virus-induced bud union incompatibility.It is known that CTLV is widespread in China,and it has caused tremendous economic loss in Zhejiang,Hunan, Fujian and Guangxi.Biological indexing is a traditional and reliable method to detect CTLV,but it is time-consuming and requires specialized facilities such as green house and screen house.So it is essential to develop a rapid and reliable method to detect CTLV,and to analyze genetic diversities of CTLV in different citrus cultivars.In this study,conventional reverse transcription-polymerase chain reaction(RT-PCR) and real-time RT-PCR detecting systems of CTLV have been established.Then restriction fragment length polymorphism(RFLP),single-strand conformation polymorphism(SSCP) and sequence analysis were applied to analyze the variation of HH,XLB and their 16 sub-isolates.The results can be concluded as follows:1.Based on a pair of primers CTL5607 and ASCT-3',a conventional RT-PCR system has been established to detect CTLV,and the amplified target band is 889 bp.2.Using a pair of primers ASG-Pf and ASG-Pr,a real-time RT-PCR system has been established to detect CTLV.It's the first time study on real-time RT-PCR has based on SYBR Green I dye for CTLV detection in China.The system to detect CTLV has high specificity (specific from Citrus tristeza virus[CTV],Satsuma dwarf virus[SDV]and Citrus psorosis virus[CPV]),great sensitivity(it is 100 times more sensitive than conventional RT-PCR) and broad applicability(it can detect CTLV isolates from different kinds of citrus cultivars),and it has several advantages such as low contamination risks,fast and easily handling.3.Eight CTLV sub-isolates were obtained by inoculating HH to eight citrus cultivars(Jincheng, lemon,Ponkan,Tangerine,Daidai sour orange,None,Fragrant orange and Rusk citrange).The RFLP patterns of coat protein gene(CP) digested by Hinf I of the eight sub-isolates and isolate HH were similar,but in the SSCP analysis of the PCR products,two to four bands were detected,indicating that isolate HH exists as a mixture of sequence variants.In sequence analysis,it was found that the nucleotide identity in the CP of the eight sub-isolates and isolate HH was very high(99.3%-100%).4.Eight CTLV sub-isolates were obtained by inoculating XLB to eight citrus cultivars(Jincheng, Lemon,Ponkan,Tangerine,Daidai sour orange,None,Fragrant orange and Rusk citrange).The RFLP patterns of CP digested by Hinf I of the eight sub-isolates and isolate XLB were similar, and in the SSCP analysis of the PCR products,two bands were detected.In sequence analysis, it was found that the nucleotide identity in the CP of the eight sub-isolates and isolate XLB was high(96.5%-99.8%).