A New Begomovirus Species in Association with Betasatellite Causing Tomato Leaf Curl Disease in Gandhinagar, India

In December 2012, tomato leaf curl disease (ToLCD) (2) was observed in tomato-growing areas of Gandhinagar District of Gujarat, a state in northwestern India. Incidence of ToLCD was estimated to be between 40 and 70% depending on the cultivars used. Infected plants exhibited symptoms consisting of leaf rolling, leaf curling, and yellowing typical of begomoviruses. Total DNA was isolated from a single affected tomato plant (2). Begomovirus infection in this sample was established by amplification of the expected-size 550-bp DNA fragment from this extract by PCR with degenerate DNA-A primers (3). Rolling circle amplification (RCA) using ϕ29 DNA polymerase was carried out on the total DNA, followed by digestion with Bam HI. An amplicon of ~2.8 kb was gel-eluted and cloned into Bam HI linearized pBluescript II KS(+). Restriction enzyme digestion of plasmid DNA from the resulting clones indicated the presence of one type of molecule. Using PCR and universal betasatellite primers, the expected 1.3-kb fragment was amplified from the DNA extract (1). An amplicon of ~1.3 kb was gel-eluted and cloned into pTZ57RT vector. Sequence analysis revealed that DNA-A (GenBank Accession No. KC952005) is composed of 2,753 nt and showed the highest identity (87.8%) with Tomato leaf curl Kerala virus[India:Kerala:2008] (GenBank Accession No. EU910141). An analysis for recombination showed this begomovirus DNA likely to have originated by recombination between Tomato leaf curl Kerala virus and Tomato leaf curl Karnataka virus. The satellite DNA-β (GenBank Accession No. KC952006) is composed of 1,365 nt and showed the highest identity (75.6%) with Tomato leaf curl betasatellite[India:Ludhiana:2004] (ToLCB-[IN:Lud:04]) (GenBank Accession No. AY765255). On the basis of DNA-A sequence analysis, the ICTV species demarcation criteria of 89% DNA-A sequence identity, and genome organization, the present isolate was considered as a new begomovirus species and named Tomato leaf curl Gandhinagar virus (ToLCGNV). The betasatellite shares less than 78% identity with (ToLCB-[IN:Lud:04]), it is considered a new species of betasatellite and the name, Tomato leaf curl Gandhinagar betasatellite (ToLCGNB) is proposed. Multimeric clones of the begomovirus and betasatellite DNAs were generated in a binary vector and these plasmids transformed into Agrobacterium tumefaciens. Nicotiana benthamiana and tomato plants agroinoculated with the cloned begomovirus DNA developed leaf curl symptoms, whereas plants co-agroinoculated with the cloned begomovirus and betasatellites developed more severe symptoms, including leaf rolling, leaf curling, and yellowing. The symptoms induced by the begomovirus and betasatellite DNAs were indistinguishable from those observed in the field. Thus, ToLCGNV is a new monopartite begomovirus which, in association with a new species of betasatellite, causes ToLCD in Gandhinagar, India. The presence of ToLCGNV needs to be considered, along with the already reported begomoviruses infecting tomatoes in this state, e.g., Tomato leaf curl Gujarat virus (2), in studies aimed to developing tomato cultivars with stable resistance to these tomato-infecting begomoviruses in India. References: (1) R. W. Briddon et al. Mol. Biotechnol. 20:315, 2002. (2) C. Reddy et al. Arch Virol. 150:845, 2005. (3) S. D. Wyatt and J. K. Brown. Phytopathology 86:1288, 1996.