Studies on the Citrus Tristeza Virus and Citrus Viroids in Pakistan

Citrus tristeza virus (CTV) is distributed worldwide, and is the causal agent of one of the most economically important and destructive diseases of citrus.Direct tissue blot immuno-assay (DTBIA) and reverse transcription polymerase chain reaction (RT-PCR) were used to pre-select CTV isolates. CTV-infected samples were then preserved in the sweet orange by graft-inoculation. The target sequences of the major coat protection gene (p25 gene) were amplified from the CTV-infected samples using RT-PCR, and the p25 genes of these isolates were analyzed by RFLP and SSCP for investigating the p25/Hinf I RFLP groups. Genetic differences and genetic relationships and the sequences of 4 genomic regions p23, p20, p18, and p25 of the 21 CTV isolates were amplified and sequenced using RT-PCR and with a sequencer. Using sequence analysis, the differences were therefore estimated among the CTV isolates from cultivated citrus. Citrus exocortis viroid (CEVd), Citrus bent leaf viroid (CBLVd), Hop stunt viroid (HSVd), Citrus dwarfing viroid (CDVd), and Citrus bark cracking viroid (CBCVd) were detected by one-step multiplex RT-PCR assay simultaneously.