B. J. Pudashini, Department of International Agricultural Development, Tokyo University of Agriculture, 1-1-1, Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan;
M. S. Shahid, Department of International Agricultural Development, Tokyo University of Agriculture, 1-1-1, Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan and Department of Biosciences, COMSATS Institute of Information Technology, Sahiwal 57000, Pakistan; and
K. T. Natsuaki, Department of International Agricultural Development, Tokyo University of Agriculture, 1-1-1, Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan
Bean common mosaic necrosis virus (BCMNV; Family Potyviridae, genus Potyvirus) infects legume crops in many regions of the world. It is transmitted in a non-persistent manner by aphids and is also readily seed-transmitted (3). Sweet bean (Lablab purpureus L.) is an important legume crop widely cultivated in Nepal. In December 2010, sweet bean plants with mottle and leaf deformation, severe mosaic, necrosis, malformation of leaves and pods, downward curling of leaves, and reduction in leaf size were observed in 20 different fields with 60 to 70% incidence in Nepal. ELISA was performed by using a universal Potyvirus antiserum test kit (Agdia Inc., Elkhart, IN) on 18 symptomatic leaf samples collected from five different locations (Malepatan, Lake side Fewataal, Darai village, Pakaudi, and Rampur) of Pokhara and Chitwan provinces and 15 out of 18 samples had a positive reaction. Filamentous shaped particles similar to Potyvirus of about 690 to 720 × 10 to 12 nm were observed by electron microscopy confirming the ELISA results. To further characterize the viral isolate(s), primary leaves of some legume crops (Phaseolus vulgaris, Glycine max, Psophocarpus tetragonolobus, Vigna unguiculata, and Arachis hypogeae) and sweet bean were mechanically inoculated with sap prepared from the same leaves used for ELISA. Phaseolus vulgaris cv. Aron and G. max cv. Togenkyou plants showed necrotic spots on inoculated leaves followed by systemic necrosis and death. Psophocarpus tetragonolobus and V. unguiculata showed systemic mosaic symptoms, while A. hypogeae and sweet bean cv. Shirobhanafuji-mame showed necrotic spots and restricted veinal necrosis. Chenopodium amaranticolor and C. quinoa also showed chlorotic local lesions on inoculated leaves. For molecular identification, total RNA was isolated from 18 symptomatic plants using Trizol Reagent (Invitrogen, Carlsbad, CA). Reverse transcription (RT)-PCR was carried out using universal primer pairs that amplify the NIb-coat protein (CP) region including the 3′-untranslated regions (UTRs) of Potyvirus as described previously (1). An amplicon of approximately 1.7 kb was amplified and cloned using the pGEM-T Easy vector system (Promega, Fitchburg, WI). Two clones (GenBank Accession Nos. AB734777 and AB735585) with 99.9% sequence identity were selected for further analysis. These clones shared a maximum of 94% amino acid identity and 90% nucleotide identity in the CP region, and 93% nucleotide homology in the 3′-UTR with the ‘TN1’ (GenBank Accession No. U37076) strain of BCMNV isolated from Phaseolus vulgaris (2). These comparisons indicated that the viral isolates belong to the BCMNV species and are the causal agent of mosaic and necrosis observed on the sweet bean plants in Nepal. To our knowledge, this is the first report of BCMNV in Nepal and also the first report of BCMNV from sweet bean.
References: (1) P. H. Berger et al. Arch. Virol. 142:1979, 1997. (2) J. Chen et al. Arch. Virol. 146:757, 2001. (3) Z. Hongying et al. Arch. Virol. 147:1257, 2002.