Chilli, which encompasses several species in the genus Capsicum, is widely consumed throughout the world. In the Indian subcontinent, production of chilli is constrained due to chilli leaf curl disease (ChiLCD) caused by begomoviruses. Despite the considerable economic consequences of ChiLCD on chilli cultivation in India, there have been scant studies of the genetic diversity and structure of the begomoviruses that cause this disease. Here we report on a comprehensive survey across major chilli-growing regions in India. Analysis of samples collected in the survey indicates that ChiLCD-infected plants are associated with a complex of begomoviruses (including one previously unreported species) with a diverse group of betasatellites found in crops and weeds. The associated betasatellites neither enhanced the accumulation of the begomovirus components nor reduced the incubation period in Nicotiana benthamiana. The ChiLCD-associated begomoviruses induced mild symptoms on Capsicum spp., but both the level of helper virus that accumulated and the severity of symptoms were increased in the presence of cognate betasatellites. Interestingly, most of the begomoviruses were found to be intra-species recombinants. The betasatellites possess high nucleotide variability, and recombination among them was also evident. The nucleotide substitution rates were determined for the AV1 gene of begomoviruses (2.60610 23 substitutions site 21 year 21 ) and the bC1 gene of betasatellites [chilli leaf curl betasatellite (ChiLCB), 2.57610 24 substitution site 21 year 21 ; tomato leaf curl Bangladesh betasatellite (ToLCBDB), 5.22610 24 substitution site 21 year 21 ]. This study underscores the current understanding of Indian ChiLCD-associated begomoviruses and also demonstrates the crucial role of betasatellites in severe disease development in Capsicum spp.

Isolates of two distinct begomovirus species, the severe strain of the species Tomato leaf curl New Delhi virus (tomato leaf curl New Delhi virus-[India:New Delhi:Severe:1992]; ToLCNDV-[IN:ND:Svr:92], bipartite) and the Varanasi strain of the species Tomato leaf curl Gujarat virus (tomato leaf curl Gujarat virus-[India:Varanasi:2001]; ToLCGV-[IN:Var:01], mono/bipartite) infect tomato (Lycopersicon esculentum) and cause severe yield losses in northern India. This study investigated the infectivity properties of genomic components of these two species. Both pseudorecombinants were infectious in Nicotiana benthamiana, Nicotiana tabacum and L. esculentum. Enhanced pathogenicity was observed when DNA-A of ToLCNDV-[IN:ND:Svr:92] was trans-complemented with ToLCGV-[IN:Var:01] DNA-B, and was consistently associated with an increase in accumulation of ToLCGV-[IN:Var:01] DNA-B. Mixed infection of ToLCNDV-[IN:ND:Svr:92] and ToLCGV-[IN:Var:01] always showed extremely severe symptoms, suggesting a synergistic interaction between these two viruses. Southern blot analysis of viral DNAs from infected plants showed a significantly higher level of accumulation of both ToLCNDV components and DNA-B of ToLCGV-[IN:Var:01] with no alteration to levels of DNA-A of ToLCGV-[IN:Var:01]. Symptom development and/or higher infectivity of the supervirulent pseudorecombinants correlated with the increased levels of DNA-B accumulation. Protoplast replication assays revealed that enhanced infectivity by the pseudorecombinant occurred at the level of replication, as DNA-A of ToLCNDV-[IN:ND:Svr:92] enhanced ToLCGV-[IN:Var:01] DNA-B replication, whose accumulation was in turn increased by ToLCGV-[IN:Var:01] DNA-A. This is the first report demonstrating a virulent pseudorecombinant between two distinct species of begomoviruses that infect tomato, and is the second report on synergism between begomoviruses. The results revealed that ToLCGV-[IN:Var:01] DNA-B is capable of associating with different DNA-A components, despite having different iteron sequences.

The full-length genome of a begomovirus and its cognate DNA-beta satellite component associated with chilli leaf curl disease (ChLCD), originating from Varanasi, India, were cloned. Sequence analysis revealed that the viral genome (EF190217) is 2,750 bp and the DNA-beta satellite (EF190215) is 1,361 bp in length. Agroinoculation with partial tandem repeats of the viral genome along with the satellite induced symptoms typical of ChLCD in chilli and Nicotiana benthamiana. However, symptom expression was delayed and milder when the viral genome was agroinoculated alone in these hosts. Sequence comparisons revealed that the genome had the highest sequence identity (95%) with that of chilli leaf curl virus-PK[PK:Mul:98]. The DNA-beta satellite shared maximum sequence identity (88%) with a DNA-beta satellite associated with tomato leaf curl disease from Rajasthan (ToLCBDB-[IN:Raj:03]). These results demonstrate that ChLCD is caused by a complex consisting of the monopartite chilli leaf curl virus and a DNA-beta satellite component. This is the first experimental demonstration of Koch's postulates using cloned DNA molecules associated with chilli leaf curl disease.

BackgroundEmerging whitefly transmitted begomoviruses are major pathogens of vegetable and fibre crops throughout the world, particularly in tropical and sub-tropical regions. Mutation, pseudorecombination and recombination are driving forces for the emergence and evolution of new crop-infecting begomoviruses. Leaf curl disease of field grown radish plants was noticed in Varanasi and Pataudi region of northern India. We have identified and characterized two distinct monopartite begomoviruses and associated beta satellite DNA causing leaf curl disease of radish (Raphanus sativus) in India.ResultsWe demonstrate that RaLCD is caused by a complex of two Old World begomoviruses and their associated betasatellites. Radish leaf curl virus-Varanasi is identified as a new recombinant species, Radish leaf curl virus (RaLCV) sharing maximum nucleotide identity of 87.7% with Tomato leaf curl Bangladesh virus-[Bangladesh:2] (Accession number AF188481) while the virus causing radish leaf curl disease-Pataudi is an isolate of Croton yellow vein mosaic virus-[India] (CYVMV-IN) (Accession number AJ507777) sharing 95.8% nucleotide identity. Further, RDP analysis revealed that the RaLCV has a hybrid genome, a putative recombinant between Euphorbia leaf curl virus and Papaya leaf curl virus. Cloned DNA of either RaLCV or CYVMV induced mild leaf curl symptoms in radish plants. However, when these clones (RaLCV or CYVMV) were individually co-inoculated with their associated cloned DNA betasatellite, symptom severity and viral DNA levels were increased in radish plants and induced typical RaLCD symptoms. To further extend these studies, we carried out an investigation of the interaction of these radish-infecting begomoviruses and their associated satellite, with two tomato infecting begomoviruses (Tomato leaf curl Gujarat virus and Tomato leaf curl New Delhi virus). Both of the tomato-infecting begomoviruses showed a contrasting and differential interaction with DNA satellites, not only in the capacity to interact with these molecules but also in the modulation of symptom phenotypes by the satellites.ConclusionThis is the first report and experimental demonstration of Koch's postulate for begomoviruses associated with radish leaf curl disease. Further observations also provide direct evidence of lateral movement of weed infecting begomovirus in the cultivated crops and the present study also suggests that the exchange of betasatellites with other begomoviruses would create a new disease complex posing a serious threat to crop production.

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