Biological and Molecular Characterization of Citrus Bent Leaf Viroid

Background and aim: Citrus bent leaf viroid (CBLVd) is one of the emerging and widely distributed viroids in citrus-growing areas of the world, including Pakistan. Previously, CBLVd has been reported in Pakistan for the first time in 2009. Therefore, characterization of CBLVd is required to monitor the viroid status in the citrus orchards concerning citrus decline. Methods: Biological and molecular characterization of CBLVd was studied through biological indexing and confirmation through RT-PCR, followed by phylogenetic analysis of selected CBLVd isolates. Among four citrus cultivars viz., Kinnow (Citrus nobilis x Citrus deliciosa), Mosambi (C. sinensis), Futrell's Early (C. reticulata) and Lemon (C. medica) used as indicator plants for two transmission trials viz., graft inoculation and mechanical inoculation. Graft inoculation was more efficient than mechanical inoculation. Results: Symptoms such as mild mosaic, slight backward leaf bending, and leaf curling were observed after eight months' post-inoculation. Citrus nobilis x Citrus deliciosa, C. reticulata and C. sinensis were more sensitive to CBLVd as compared to C. medica. Inoculated plants were reconfirmed through RT-PCR amplicons of 233 bp. The phylogenetic tree of submitted sequences showed more than 90% relevance of CBLVd in Pakistan compared to the rest of the world. Conclusions: There was slight genetic variability, but more than 90% relevance was found among the submitted and already reported CBLVd isolate from Pakistan. Scanty literature is available regarding the biological and molecular studies of CBLVd in Pakistan. Therefore, the transmission and molecular characterization of CBLVd in Pakistan were studied for the first time.