S.11. Identification of diversified Citrus tristeza virus population isolates from eastern himalayan region of India by hetroduplex mobility assay and nucleotide sequence analysis

Citrus tristeza closterovirus (CTV), transmitted by the brown citrus aphid (BCA, Toxoptera citricida) is a positive-sense RNA virus with a 20 kb genome. CTV causes the most economically important citrus decline disease worldwide. Symptoms include stem pitting, seedling yellows, decline, vein clearing and vein corking, depending on the scion/rootstock combination. Four isolates ofCTV were collected from original source plant of Darjeeling mandarin (Citrus reticulata) or Kagzi lime (Citrus aurantifolia) having characteristic CTV vein clearing/vein flecking and stem pitting symptoms from four locations of Kalimpong Subdivision of Darjeeling Disgtrict in India designated as Kpg-1, Kpg-2, Kpg-3 and Kpg-4. The isolates are maintained in Kagzi lime. These four isolates were analyzed by biological indexing, ELISA with polyclonal antisera, and RT-PCR using 10 sets of oligonucleotide multiple molecular markers for genotyping, the specific primers for coat protein, and universal primers for a variable region of the CTV genome on the 5′ end. The RT-PCR amplified products from the coat protein amplifying primers and universal primers were screened by the heteroduplex mobility assay to detect genotype differences of CTV. Three groups of CTV were identified on the basis of coat protein gene sequence and two groups on the basis of the variable region on the 5′ end by molecular cloning, and nucleotide sequencing. Differences in nucleotide sequences of these CTV isolates of Kalimpong were analysed and compared with other Indian CTV and other exotic CTV available in database, and their Phylogenetic relationship was established.