A new Begomovirus species causing leaf curl disease of radish in India

Abstract

Leaf curl disease of radish (RLCD) was observed for the first time in India in commercial fields and kitchen gardens of the Varanasi District and adjoining areas of eastern Uttar Pradesh during November 2003. Infected plants exhibited typical upward and downward leaf curling, leaf distortion, reduction of leaf area, and conspicuous enations on the underside of the leaves. Incidence of RLCD was estimated to be between 10 and 40% depending on the cultivars used. Electron microscopic observation revealed typical geminate particles in infected leaf samples. The causal virus could be transmitted to radish cv. Minu Early by whiteflies (Bemisia tabaci) and grafting. Inoculated plants developed symptoms similar to those observed in naturally infected radish plants. Viral DNA was isolated from artificially inoculated symptomatic radish plants (4) followed by concentration of super-coiled DNA by alkaline denaturation (1). The presence of a geminivirus was confirmed by PCR using DNA-A degenerate primers (3), and a 1.5-kb amplified product was obtained from six artificially and three naturally infected plants. Amplification of the full-length DNA-A was achieved using a primer combination derived from sequences obtained from a 1.5-kb amplicon. Amplification of 1.3-kb DNA-β sequences was achieved using specific primers (2) in three infected plants. Sequence analysis revealed that DNA-A (GenBank Accession No. EF 175733) contained 2,756 nt and DNA-β contained 1,358 nt (GenBank Accession No. EF 175734). DNA-A of the causal virus shares 87.7% identity with Tomato leaf curl Bangladesh virus (GenBank Accession No. AF 188481) and 62% identity with Mungbean yellow mosaic India virus (GenBank Accession No. AF126406). The begomovirus DNA-A sequence associated with RLCD contained seven open reading frames (AV1, AV2, AC1, AC2, AC3, AC4, and AC5). The DNA-β associated with RLCD shared the highest nucleotide sequence identity (84.9%) with DNA-β of Tobacco leaf curl virus isolate NIB 12-1 (GenBank Accession No. AJ316033) reported from Pakistan. Despite exhaustive attempts to amplify a putative viral B-component using degenerate primers based on the intergenic region sequence of the DNA-A or sequences that are highly conserved for other begomoviruses, no DNA-B component was detected. On the basis of DNA-A sequence analysis, the ICTV species demarcation criteria of 89% sequence identity, and genome organization, the virus causing RLCD should be considered a new Begomovirus species, for which the name Radish leaf curl virus (RLCV) is proposed. To our knowledge, this is the first report of the association of a Begomovirus with a disease of radishes in India