Pea (Pisum sativum) plants exhibiting leaf distortion, yellowing, stunted growth and reduction in leaf size from Rampur, Nepal were shown to be infected by a begomovirus in association with betasatellites and alphasatellites. The begomovirus associated with the disease showed only low levels of nucleotide sequence identity (<91%) to previously characterized begomoviruses. This finding indicates that the pea samples were infected with an as yet undescribed begomovirus for which the name Pea leaf distortion virus (PLDV) is proposed. Two species of betasatellite were identified in association with PLDV. One group of sequences had high (>78%) nucleotide sequence identity to isolates of Ludwigia leaf distortion betasatellite (LuLDB), and the second group had less than 78% to all other betasatellite sequences. This showed PLDV to be associated with either LuLDB or a previously undescribed betasatellite for which the name Pea leaf distortion betasatellite is proposed. Two types of alphasatellites were identified in the PLDV-infected pea plants. The first type showed high levels of sequence identity to Ageratum yellow vein alphasatellite, and the second type showed high levels of identity to isolates of Sida yellow vein China alphasatellite. These are the first begomovirus, betasatellites and alphasatellites isolated from pea.
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.
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