The Genetic Diversity Of Citrus Tristeza Virus

Citrus tristeza disease caused by Citrus tristeza virus (CTV) is one of the most destructive and economically important diseases of commercial citrus worldwide. It mainly causes losses of sweet orange trees on sour orange rootstock, and also causes stem pitting syndrome and reduces fruit size of grapefruits and some sweet orange varieties. In this study, RT-PCR, SSCP, DNA sequencing and phylogenetic analysis are used to investigate the CTV infection of citrus in China, to compare sequence characteristics of different isolates, to explore their phylogenetic relationships, and give theoretical guides in developing molecular markers to finally identify the CTV strains in China. The results are described as following:1. 242 samples of satsuma mandarin (Citrus unshiu Marc), tangerine (C. reticulata Blanc), sweet orange (C. sinensis Osb.) and pummelo (C. grandis Osb.) were randomly collected from ten counties of Hubei, Hunan, and Jiangxi. CP (Coating protein) genes of CTV from the samples preliminarily screened by PAS-EL1SA were amplified by RT-PCR. The amplification results showed that 120 samples from fourteen sampling sites were infected with CTV, the infecting rate of single site ranged from 20.0% to 71.4%, and the total rate was up to 49.6%. Thus it indicated that CTV extensively existed in these regions. Single-strand conformation polymorphism (SSCP) analysis showed that 120 amplified products of CP gene made up a profile of 120 kinds of SSCP patterns and each pattern consisted of two to seven single-strand DNA bands. This suggested that a great mixed infection of CTV existed among these sampling regions.The total RNA extracted by a method of CTAB-LiCl could always work as efficient templates in RT-PCR detection of CTV in citrus, and its preparation was simple and not expensive.2. CP genes of CTV were amplified by RT-PCR with total RNA as templates extracted from three tangerine seedling samples (HN, JX-1 and JX-2) collected from Daoxian, Hunan and Chongyi, Jiangxi. The results of cloning and sequencing of amplified products demonstrated that three samples were infected with CTV. The SSCP analysis results before and after cloning the amplified products showed that the CP genes from HN and JX-1 only contained a type of unique sequence, but JX-2 had a population of sequences, two of which were predominant. This indicated that genetic variation existed among the CP genes of CTV from three samples, and JX-2 was heterogeneous probably due to the mixed infection.Bi-directional sequencing and sequence analysis showed that four CP gene sequences had full-length translating regions without any base insertion, deletion, or stop code insertion. Nucleotide sequence alignment results indicated that four clones shared 93%-99% of nucleotide sequence similarity. Clone HN-K had 98% of similarity with Portuguese isolate 28C and Israel stem pitting isolate VT, clone JX-1-1 had 98-99% with Indian isolate Pune and Bangalore, and clone JX-2-7 and JX-2-17 had over 97% with Japanese seedling yellows...