Diagnostic assays for two closely related tospovirus species, <Emphasis Type="Italic">Watermelon bud necrosis virus</Emphasis> and <Emphasis Type="Italic">Groundnut bud necrosis virus</Emphasis> and identification of new natural hosts

Watermelon bud necrosis virus (WBNV) and Groundnut bud necrosis virus (GBNV) are two closely related tospovirus species infecting several crops in India. In the present study, specific diagnostic assays for these two most predominant tospoviruses were developed by comparing host reactions and genome sequence information. Bioassay was developed by sap transmission of both the viruses to a set of host plants, cowpea, groundnut and watermelon. WBNV induced only local symptoms including chlorotic spots on cowpea cv. Pusa Komal, typical chlorotic ring spots on cv. C-152, bud necrosis on watermelon and no symptoms on groundnut. Whereas, GBNV induced both local and systemic symptoms including chlorotic/necrotic spots and veinal necrosis on both the cowpea cultivars, systemic yellowing and bud necrosis on groundnut and no symptoms on watermelon. Single and duplex RT-PCR was developed for diagnosis of WBNV and GBNV by designing primers based on differential sequence from Gn/Gc genes located on M RNA and from nucleocapsid protein (NP) gene located on S RNA genome. The duplex RT-PCR using NP gene specific primers was successfully utilised for the diagnosis of natural infection of GBNV in dahlia and muskmelon and mixed infection of GBNV and WBNV in chrysanthemum. The amplified products of duplex RT-PCR were sequenced and phylogenetic analysis confirmed the authenticity of the RT-PCR assay as well as confirmed infection of WBNV and GBNV in the new natural hosts. The bioassay and RT-PCR developed in this study will be useful in detecting single and mixed infection of GBNV and WBNV strains in different crops.