Pakistan is among the top 10 citrus-producing countries of the world and the leader in Kinnow mandarin production with production concentrated in the province of Punjab, which produces more than 96% of the total citrus crop. To evaluate the presence and distribution of citrus viroids in this area, 34 samples were collected in September 2008 from citrus orchards in the Sargodha, Bhalwal, and Faisalabad areas of Punjab, including 15 ‘Mosambi’ and two ‘Bloodred’ sweet oranges (Citrus sinensis), eight ‘Kinnow’ and four ‘Feutrell Early’ mandarins (C. reticulata), three ‘Jatti Khatti’ rough lemon (C. jambhiri), and two grapefruit (C. paradisi), which showed stunting, bark scaling, and cracking symptoms on the rootstock which was either citrange (Poncirus trifoliata × C. sinensis) or sweet lime (C. limetta). Infected budwood from these trees was grafted onto indicator plants of Arizona 861-S-1 ‘Etrog citron’ (C. medica) budded on rough lemon rootstock, and after 3 months, the citron showed typical viroid symptoms of mild epinasty and leaf roll with 23 of the 34 samples. A one-step multiplex reverse transcription (RT)-PCR assay (3) was used to detect simultaneously Citrus exocortis viroid (CEVd), Citrus bent leaf viroid (CBLVd), Hop stunt viroid (HSVd), Citrus dwarfing viroid (CDVd), and Citrus bark cracking viroid (CBCVd). On the basis of amplification of the appropriately sized DNA, CEVd, CBLVd, HSVd, and CDVd were detected in 12, 8, 31, and 17 samples, respectively, whereas CBCVd was not detected. Twenty-three of 34 infected samples harbored more than one viroid species and one had four viroids. Budwood from 11 trees did not induce viroid symptoms on Etrog citron. Two of these trees were infected with CBLVd only and nine with HSVd only. Four primer pairs were used to amplify the full sequences of CEVd, CBLVd, HSVd, and CDVd by RT-PCR (2), which were cloned by standard methods. Sequences of three cDNA clones each of CEVd (Nos. FJ773253, FJ773254, and FJ773255), CBLVd (Nos. FJ773262, FJ773263, and FJ773267), HSVd (Nos. FJ773268, FJ773269, and FJ773271), and CDVd (Nos. FJ773274, FJ773275, and FJ773276) were deposited in GenBank. BLAST analysis showed that these nucleotide sequences had greater than 97% nucleotide identity to the most similar genome sequences in GenBank. One of the HSVd sequences, FJ773271, presented the cachexia determinants. To our knowledge, this is the first report of CBLVd and CDVd in Pakistan (1). These results indicate the need for proper indexing of mother trees and a virus-free propagation scheme to create healthy budwood sources in Pakistan. References: (1) M. Arif et al. Pak. J. Bot. 37:407, 2005. (2) L. Bernard and N. Duran-Vila. Mol. Cell. Probes 20:105, 2006. (3) X. F. Wang et al. Eur. J. Plant. Pathol, 124:175, 2009.
