First report of Tomato leaf curl Gujarat virus, a bipartite begomovirus on cotton showing leaf curl symptoms in Pakistan

Abstract

Cotton leaf curl is one of the most important constraints on cotton (Gossypium hirsutum) in Pakistan and northwestern India. The disease is associated with several monopartite begomoviruses, which essentially require a specific DNA satellite called Cotton leaf curl Multan betasatellite. The disease complex is evolving rapidly by component capture and recombination, which supposedly helps the complex to overcome resistance (Sattar et al. 2013). During a routine survey for monitoring cotton leaf curl disease in October 2013, several leaf samples of infected cotton plants showing typical symptoms of disease such as leaf curling and vein thickening were collected from the Gojra region of Punjab, and were checked for the presence of begomoviruses and associated satellites. Total DNA extracted from infected plants was resolved on agarose gel, transferred to nylon membrane, and probed with Cotton leaf curl Burewala virus probe. The probe detected begomovirus DNA forms found in two out of six infected plants and confirmed the association of begomovirus with the disease. Universal primers for begomoviruses (Akhtar et al. 2009) were used in PCR for confirmation of begomoviruses, and PCR products of expected size (∼2.8 kb) were obtained. For the detection of begomoviruses, the amplified products were cloned in a TA cloning vector and were sequenced. The complete nucleotide sequences of two clones showed 99% sequence identity with Tomato leaf curl Gujarat virus (ToLCGuV), available in database under accession numbers LN794214 and LN794215, a bipartite begomovirus reported from India (Chakraborty et al. 2003). ToLCGuV was previously found on a weed species in Pakistan but effort to find cognate DNA B was not successful (Mubin et al. 2012). In order to confirm the bipartite nature of the virus, specific primers were designed on DNA B (forward primer 5′-GGTACCCGTAACGATCTTGAACTATGTCCC-3′ and reverse primer 5′-GGTACCCTATCTGGCTATAGGTCCGAACG-3′). The use of these primers in PCR resulted in amplification of a full length ∼2.7 kb product, confirming the bipartite nature of virus. PCR analysis of 40 plant samples was done, among which 14 showed amplification of expected size. The amplified product was cloned and fully sequenced. The sequence analysis confirmed the identity as DNA B of Tomato leaf curl New Delhi virus (ToLCNDV), available in database under accession number LN713269, which is 92% identical to the ToLCNDV DNA B isolated from Sonchus arvensis, a weed found in Pakistan (Mubin et al. 2010). To our knowledge, this is the first report of a bipartite begomovirus on cotton showing leaf curl disease symptoms; however, further work is needed to confirm Koch’s postulate on cotton. The association of a bipartite virus may help the virus complex to overcome host disease resistance or expansion of the host range.