Study On Serological And Molecular Characteristics Of Citrus Tristeza Virus

Citrus tristeza virus (CTV) causes one of the most economically important citrus diseases in worldwide. The genome of CTV is changed as adjustment of citrus cultivation structure and new breed, lead to the pathogenicity, symptoms, and the damage on citrus are harder than before. In this study, the characteristic about serology, biology and molecular biology of CTV from some samples in china citrus cultivation were as follows:All of 111 samples were from3 provinces (Hubei, Jiangxi and Sichuan) and detected by 10 CTV monoclonial antibodies (MCAs) with DAS-Ⅰ-ELISA. CTV was widely distributed in these areas and its positive value was more than 60%. 30 CTV positive samples were chosed as distinct biological difference and symptoms on field citrus. Different interaction was existed in the same sample detected with different MCAs. Also, difference was found in the different sample detected by the same MCAs. According to the amount of MCAs which could not test the same sample with DAS-I-ELISA, 30 CTV positive samples were divided into ten types.Thirty positive samples were grafted on four species indicators: Citrus aurantium, C. limetta, C. medica, C. paradise. After 3 months, typical symptoms of CTV caused were showed on indicators or buds. On Mexican lime and sweet orange, CTV induced stem pitting and chlorotic.CTV caused quickly decline, leaves cupped, vein lignification, shot leaves yellowing partially, shoot aging, on sour orange. On sweet orange on sour orange rootstock, CTV induced fruit malformation, sheding leaves, top shoot withered.The coat protein (CP) gene of CTV for 30 samples was amplified using CP specific primers, yielding an amplicon of 672 bp for all isolates. N3 and N4 were infected by CTV mild isolates, had a typical 392 bp band which was amplified with specific primer of isolates in the agarose gel. CTV from the other 28 samples had a 320 bp band, were infected by severe isolates. In analysis of CTV CP gene by HinfⅠ/RFLP, N3 and N4 were groupⅣ, N25 was groupⅥ, N21 was a new group and defined as groupⅧ, as reported.Four isolates (N25, N3, N4 and N21) were infected by one group, the other samples were mixed infection. N25, N3, N4 and N21 are nominate as CTV-N25, CTV-N3, CTV-N4 and CTV-N21.CP genes of four CTV isolates (CTV-N25, CTV-N3, CTV-N4, and CTV-N21) were cloned and sequenced. The CP gene nucleotide sequences and coat proteinamino acid identities of of CTV-N3 and CTV-N4 were similar to the mild isolate T30(＞99%). Amino acids at the site from123aa to 125aa of CTV-N3 and CTV-N4 wereⅣY the same as the special feature amino acid of mild isolates, as Pappu et al.(1993) reported .So, CTV-N3 and CTV-N4 were mild isolates. The nucleotide sequences identities between CTV-N21 and T318A (severe isolate) was 95.8%, CTV-N25 and severe mexico isolate was 96.4%. The CTV-N21 and CTV-N25 showed highest coat protein aa identity with SY568 96.4% and 97.3%, respectively. Amino acids at the site from123aa to 125aa of CTV-N21 and CTV-N25 were VVF the same as the special feature amino acid of severe isolates, as Pappu et al. (1993) reported.Therefore, CTV-N21 and CTV-N25 were belong to severe isolates. CP gene of mild isolates had a consistent variability with severe isolates: some sites of CP gene are gene replacement, only three sites are transversion (T←→A).The nucleotide sequence data reported in this paper have been deposited in the GenBank database under accession numbers: CTV-N3 (EF517334), CTV-N4 (EF517335), CTV-N21 (EF063109), CTV-N25 (EF5173366).