ABSTRACT
ABSTRACT The biological and molecular properties of Tomato leaf curl Gujarat virus from Varanasi, India (ToLCGV-[Var]) were characterized. ToLCGV-[Var] could be transmitted by grafting and through whitefly transmission in a persistent manner. The full-length genome of DNA-A and DNA-B of ToLCGV-[Var] was cloned in pUC18. Sequence analysis revealed that DNA-A (AY190290) is 2,757 bp and DNA-B (AY190291) is 2,688 bp in length. ToLCGV-[Var] could infect and cause symptoms in tomato, pepper, Nicotiana benthamiana, and N. tabacum when partial tandem dimeric constructs of DNA-A and DNA-B were co-inoculated by particle bombardment. DNA-A alone also is infectious, but symptoms were milder and took longer to develop. ToLCGV-Var virus can be transmitted through sap inoculation from infected tomato plants to the above-mentioned hosts causing the same symptoms. Open reading frames (ORFs) in both DNA-A and DNA-B are organized similarly to those in other begomoviruses. DNA-A and DNA-B share a common region of 155 bp with only 60% sequence identity. DNA-B of ToLCGV-[Var] shares overall 80% identity with DNA-B of Tomato leaf curl New Delhi virus-Severe (ToLCNDV-Svr) and 75% with ToLCNDV-[Lucknow] (ToLCNDV-[Luc]). Comparison of DNA-A sequence with different begomoviruses indicates that ToLCGV-[Var] shares 84% identity with Tomato leaf curl Karnataka virus (ToLCKV) and 66% with ToLCNDV-Svr. ToLCGV-[Var] shares a maximum of 98% identity with another isolate of the same region (ToLCGV-[Mir]; AF449999) and 97% identity with one isolate from Gujarat (ToLCGV-[Vad]; AF413671). All three viruses belong to the same species that is distinct from all the other geminivirus species described so far in the genus Begomovirus of the family Geminiviridae. The name Tomato leaf curl Gujarat virus is proposed because the first sequence was taken from an isolate of Gujarat, India.
ABSTRACT
In November 2001, a leaf curl disease of tomato, manifested by yellowing of leaf lamina, upward leaf curling, leaf distortion, shrinking of leaf surface, and stunted plant growth was observed in tomato-growing areas in the Varanasi and Mirzapur districts of eastern Uttar Pradesh, India, which caused yield losses up to 100%. The causal agent was infective to tomato cv. Punjab Chuhara by whiteflies and grafting. Inoculated plants developed symptoms observed in naturally infected tomatoes. Viral DNA was isolated from artificially inoculated tomato plants using 1% CTAB (2) followed by a concentration of supercoiled DNA by alkaline denaturation (1). A geminivirus was confirmed by polymerase chain reaction using DNA-A degenerate primers (3), and a 550-bp amplified product was obtained from artificially and naturally infected plants. Full-length viral genomes of DNA-A and DNA-B were cloned in plasmid pUC18 at HindIII and XbaI sites, respectively. Partial tandem dimers of the viral clones were infective to Nicotiana benthamiana and tomato cv. Organ Spring through particle bombardment. Infected N. benthamiana plants exhibited downward and upward leaf curling, big veins, leaf puckering with interveinal chlorosis, and stunting. On tomato, symptoms were the same as those seen on naturally infected plants. Cloned DNA also infected Capsicum annuum cv. California Wonder (upward leaf curling and stunting) and tobacco cv. Xanthi (leaf curling and crinkling) but failed to infect Phaseolus vulgaris, okra, cotton, and N. glutinosa. The Varanasi isolate was sap transmissible (0.1 M potassium phosphate buffer, pH 7.0) from the bombarded plants to N. benthamiana and tomato cv. Organ Spring. DNA-A alone infected N. benthamiana (upward leaf curling and big veins) and tomato cv. Organ Spring (mild leaf curl), but symptoms were delayed and milder. Full-length genome sequencing revealed DNA-A (AY190290) contained 2,757 nt and DNA-B (AY190291) contained 2,688 nt. DNA-A of the Varanasi isolate shares 98.4% identity with a DNA-A sequence (AF449999) obtained from a tomato showing leaf curl symptoms from the same region and 97.1% identity with an isolate from Gujarat (900 km from Varanasi). All three sequences represent isolates of the same species, herein called Tomato leaf curl Gujarat virus, based on the priority of submission of the DNA sequence for the Gujarat region (ToLCGV; AF 413671). All isolates noted were obtained from GenBank. However, except for the DNA-A sequence, no other information is available for these ToLCGV isolates. DNA-A of the ToLCGV-Varanasi isolate shares 66.8% identity with Tomato leaf curl New Delhi virus, severe strain (ToLCNdV-Svr) (U15015), and 84.1% with Tomato leaf curl Karnataka virus (U38239). No DNA-B has been reported for these two ToLCGV isolates, and no infectious clone proving the etiology of the disease has been constructed, except for ToLCGV-Varanasi. DNA-B of ToLCGV-Varanasi shares 79.2% homology with ToLCNdV-Svr and 84.1% with ToLCNdV-Luc (X89653). These results suggest that the isolate from Varanasi belongs to ToLCGV, a previously undescribed geminivirus species causing a devastating tomato leaf curl disease in Gujarat and Uttar Pradesh. References: (1) H. C. Birnboim and J. Doly. Nucleic Acids Res. 7:1513, 1979. (2) K. M. Srivastava et al. J. Virol. Methods 51:297, 1995. (3) S. D. Wyatt and J. K. Brown. Phytopathology 86:1288, 1996.
ABSTRACT
The Asian Vegetable Research and Development Center's (AVRDC) tomato breeding lines derived from Lycopersicon hirsutum f. glabratum B 6013 × L. esculentum H-24 and carrying the Ty-2 resistance gene located on chromosome 11 are tolerant to tomato leaf curl disease in Karnataka State, southern India (3), where several isolates of Tomato leaf curl Virus-Bangalore (GenBank Accession Nos. L11746, Z48182, and AF165098) and Tomato leaf curl virus-Karnataka (GenBank Accession No. U38239) are reported to infect tomatoes. The only area in south and southeast Asia where these AVRDC tomato breeding lines were found susceptible to begomovirus infection is Thailand, where several bipartite Tomato yellow leaf curl virus isolates (GenBank Accession Nos. X63015, X63016; AF141922, AF141897; and AF511529, AF511528) are reported to be prevalent. However, in field trials conducted in the fall of 1999 in Bodeli, Gujarat State, western India, the AVRDC breeding lines showed typical symptoms of begomovirus infection, such as leaf curling and vein clearing. The presence of a different tomato begomovirus was suspected. Viral DNA from a symptomatic plant from Bodeli was amplified by polymerase chain reaction (PCR) using the begomovirus-specific degenerate primer pair PAL1v1978/PAR1c715 (4) and the expected 1.4-kb PCR product was obtained. Based on the sequence of the 1.4-kb DNA product, specific primers were designed to complete the DNA-A sequence. The DNA-A of the virus associated with tomato leaf curl from Bodeli consists of 2,759 nucleotides (GenBank Accession No. AF413671) and contains six open reading frames (ORFs V1, V2, C1, C2, C3, and C4). The DNA-A sequence of the Bodeli isolate had highest sequence identities of 98 and 98.3%, respectively, with viruses causing tomato leaf curl from Varanasi, Uttar Pradesh State, northern India (GenBank Accession No. AF449999) collected in the fall of 1999 and Panchkhal, Nepal (GenBank Accession No. AY234383) collected in early 2000. There was no evidence for the presence of DNA-B in the Bodeli, Panchkhal, or Varanasi virus isolates using DNA-B specific primer pairs DNABLC1/DNABLV2 and DNABLC2/DNABLV2 (2). However, a 1.3-kb DNA-beta was detected in the Panchkhal and Varanasi isolates using the primer pair Beta01/Beta02 (1). Sequence comparisons with begomovirus sequences available in the GenBank database showed that these three virus isolates and GenBank Accession No. AY190290 collected in 2001 from Varanasi shared more than 97% sequence identity with each other and should be considered closely related strains of the same virus. These four virus isolates belong to a new distinct tomato geminivirus species because their sequences share less than 88% sequence identities with the next most closely related virus, Tomato leaf curl virus-Karnataka (GenBank Accession No. U38239). This new tomato leaf curl virus is prevalent in western India, northern India, and Nepal. References: (1) R. W. Briddon et al. Mol. Biotechnol. 20:315, 2002. (2) S. K. Green et al. Plant Dis. 85:1286, 2001. (3) V. Muniyappa et al. HortScience 37:603, 2002. (4) M. R. Rojas et al. Plant Dis. 77:340, 1993.
