Gaurav Raikhy

Molecular studies on Tomato aspermy virus isolates infecting chrysanthemums

Abstract Tomato aspermy virus (TAV), an important viral pathogen of chrysanthemums, was detected by double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA) from chrysanthemums exhibiting mottling and deformed inflorescence grown in various states of India. Out of 15 cultivars (cvs.) tested, 11 (73.3%) were found to be positive for TAV. On sap inoculation the virus produced local lesions on Chenopodium spp. and Cucumis sativus. Nicotiana clevelandii, N. glutinosa, N. megalosiphon and N. tabacum reacted systemically to the virus producing severe mosaic, leaf deformation and characteristic leaf enations. Tomato plants produced malformed fruits (2-3/plant) with a few seeds. Myzus persicae and Aphis gossypii transmitted the virus non-persistently. Electron microscopy of partially purified viral preparations revealed polyhedral virions (ca. 29 nm dia). Cytopathology of infected leaves of N. clevelandii showed crystalline inclusions containing virions in the central vacuole of infected cells. The virions showed very good clumping with antiserum to TAV in liquid phase immuno-electron microscopy. Slot blot hybridization was performed to detect the virus in various chrysanthemum cultivars. The virus positivity was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and nucleotide sequencing of coat protein gene (CP). The amplified 657 bp fragment was about 97% identical with respect to CP sequences of other TAV isolates available in the database. In terms of derived amino acid sequence similarity, the homology value was 99%. In order to amplify RNA 1, RNA 2 and RNA 3, multiplex RT-PCR was performed. High genetic similarities of CP gene of TAV Indian isolates with that of other isolates of TAV indicate their probable common ancestry.

Potential uses of in vitro expressed and purified recombinant Prunus necrotic ringspot virus coat protein gene.

Abstract Prunus necrotic ring spot virus (PNRSV), an ilarvirus, has a wide host range especially on important commercial crops like almond, apple, apricot, hop, peach, and rose. PNRSV coat protein gene was cloned in expression vector pGEX-2Tk (Amersham Pharmecia, USA) using E. Coli BL 21 strain competent cells. Expression conditions were standardized for maximum recovery of soluble recombinant protein. The in vitro expressed protein was purified and used as an antigen for raising antisera. Both intramuscular and sub-cutaneous routes were used separately for antisera raising. A part of the raised and purified antisera was used in enzyme conjugate preparation. It was then formulated in the form of an ELISA-based diagnostic kit for PNRSV detection. It was also compared with the commercially available kit.