Direct Foliar Application of dsRNA Derived From the Full-Length Gene of NSs of Groundnut Bud Necrosis Virus Limits Virus Accumulation and Symptom Expression

Groundnut bud necrosis virus (GBNV) is the most significant member of the genus Orthotospovirus occurring in the Indian subcontinent. There is hardly any effective measure to prevent GBNV in crop plants. In order to develop GBNV infection prevention procedure, we examined the effect of the direct foliar application of double-stranded RNA (dsRNA) derived from the full-length NSs gene (1,320 nucleotides) of GBNV. The bacterially expressed dsRNA to the non-structural (dsNSs) gene of GBNV was purified and delivered to plants as an aqueous suspension containing 0.01% Celite for evaluating its efficacy in preventing GBNV infection in systemic host, Nicotiana benthamiana as well as in local lesion and systemic host, cowpea cv. Pusa Komal (Vigna unguiculata). The dsNSs application and challenge-inoculation were conducted in three different combinations, where plants were challenge-inoculated with GBNV a day after, immediately, and a day before the application of dsNSs. N. benthamiana plants, which were not treated with dsRNA showed severe systemic wilting and death by 9–16 days post-inoculation (dpi). The non-treated cowpea plants exhibited many chlorotic and necrotic lesions on the cotyledonary leaves followed by systemic necrosis and death of the plants by 14–16 dpi. The dsNSs treated plants in all the combinations showed significant reduction of disease severity index in both N. benthamiana and cowpea. The treatment combination where the GBNV inoculation was conducted immediately after the dsNSs treatment was found to be the most effective treatment in preventing symptom expression. The viral RNA analysis by real time PCR also showed 20 and 12.5 fold reduction of GBNV in cowpea and N. benthamiana, respectively. Our results suggest that the foliar application of dsRNA derived from the full-length NSs gene of GBNV through Celite is successful in delivering long dsRNA leading to effective prevention of GBNV infection.

Co-existence of chlorosis inducing strain of Cucumber mosaic virus with tospoviruses on hot pepper (Capsicum annuum) in India

Cucumo- and tospoviruses are the most destructive viruses infecting hot pepper (chilli). A diagnostic survey was conducted to assess the prevalence of cucumo and tospoviruses in chilli growing tracts of Tamil Nadu. Infected plants showing mosaic with chlorotic and necrotic rings, veinal necrosis, mosaic mottling, leaf filiformity and malformation were collected. Molecular indexing carried out through reverse transcription polymerase chain reaction (RT-PCR) with coat protein gene specific primer of Cucumber mosaic virus (CMV) and tospovirus degenerate primer corresponding to the L segment (RdRp). Ostensibly, amplifications were observed for both CMV and tospoviruses as sole as well for mixed infections. The sequence analysis indicated that the Capsicum chlorosis virus (CaCV) and Groundnut bud necrosis virus (GBNV) to be involved with CMV in causing combined infections. The co-infection of CMV with CaCV was detected in 10.41% of the symptomatic plant samples and combined infection of CMV with GBNV was recorded in around 6.25% of the symptomatic plants surveyed. The amino acid substitution of Ser129 over conserved Pro129 in coat protein of CMV implies that CMV strain involved in mixed infection as chlorosis inducing strain. Further, the electron microscopy of symptomatic plant samples explicated the presence of isometric particles of CMV and quasi spherical particles of tospoviruses. This is the first molecular evidence for the natural co-existence of chlorosis inducing CMV strain with CaCV and GBNV on hot pepper in India.

Characterization of a New Orthotospovirus from Chilli Pepper in Yunnan Province, China

Chilli pepper (Capsicum annuum L.) is one of the most important crops in Yunnan Province, China. An orthotospovirus isolate 14YV855 was isolated from a diseased chilli pepper plant exhibiting yellow ringspots and necrosis on leaves in Shiping County, Honghe Hani and Yi Autonomous Prefecture, Yunnan Province in 2014. The complete genome sequence of 14YV855 was determined. The small, medium, and large RNAs are 3,428, 4,781, and 8,917 nucleotides long, respectively. The complete nucleocapsid (N) protein of 14YV855 shares a high amino acid identity of 84.8 to 89.9% to that of Capsicum chlorosis virus (CaCV), Groundnut bud necrosis virus (GBNV), Watermelon bud necrosis virus (WBNV), and Watermelon silver mottle virus (WSMoV), which is slightly less than the 90% identity threshold for the demarcation of new Orthotospovirus sp. Phylogenetic analyses revealed that the N protein and RNA-dependent RNA polymerase of 14YV855 are the most related to WSMoV, while the NSs, NSm, and Gn/Gc proteins are similar to those of GBNV. As expected, 14YV855 is serologically related to CaCV, GBNV, WBNV, and WSMoV when the monoclonal antibody against the N protein of WSMoV was used; however, 14YV855 can be distinguished from other orthotospoviruses by reverse-transcription PCR using the specific primers. Our results indicate that 14YV855 is a new Orthotospovirus sp. belonging to the WSMoV serogroup and is provisionally named Chilli yellow ringspot virus.