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First Report of Cotton Leaf Curl Disease in Central and Southern Sindh Province in Pakistan.

Cotton leaf curl is a devastating disease of cotton that has resulted in severe losses (estimated at more than US$87 million per annum) in Pakistan. The epidemic is centered in Punjab, the province that contributes approximately 80% of Pakistan's cotton. Previously, the disease had been observed sporadically on single plants in the northern Sindh Province but did not cause economically significant damage. During the years 2004 and 2005, a high incidence (approximately 20%) of the disease was observed in Shahdadpur and parts of District Sanghar, located in central Sindh Province. The disease was also observed at low incidence (<1%) in southern Sindh. To confirm the identity of the causal agent of the disease, 18 samples from three districts in central southern Sindh (Sanghar, Hala, and Hyderabad) were collected, and total DNA was extracted using cetyltrimethylammoniumbromide (2). Universal primers for begomoviruses based on conserved sequences as follows were used in polymerase chain reaction (PCR): BegomoF (5'-CCGTGCTGCTGCCCCCATTGTCCGCGTCAC-3') and BegomoR (5'-CTGCCACAACCATGGATTCACGCACAGGG-3'). Universal primers for amplification of DNA β with PCR were also used (1). A full-length clone of Cotton leaf curl Multan virus (CLCuMV) was labeled with alpha-32PdCTP by the oligo-labeling method and used as a probe in Southern hybridization for the detection of geminivirus DNA forms (2). Similarly, cotton leaf curl disease associated DNA β was also labeled and used as a probe in Southern hybridization. The use of universal primers for begomoviruses resulted in amplification of viral DNA of the expected size from all samples while no PCR product was obtained from healthy plants. PCR results confirmed that all plants were infected with begomoviruses. Southern hybridization with CLCuMV and DNA β probes detected begomovirus DNA forms associated with virus replication when washed at medium stringency, further confirming that the plants were infected with the cotton leaf curl geminivirus complex (2). Our results indicate that cotton leaf curl complex has become established in central and southern districts of Sindh Province and it poses a major threat to cotton grown in the region. References: (1) R. W. Briddon et al. Mol. Biotechnol. 20:315, 2002. (2). S. Mansoor et al. Arch. Virol. 148:1969, 2003.