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First Report of Groundnut Bud Necrosis Virus in Pergularia daemia (Asclepiadaceae) in India

Author:
Aravintharaj, R., Asokan, R., Pavithra, B. S., Reddy, M. K.
Source:
Plant disease 2018 v.102 no.12 pp. 2671
ISSN:
0191-2917
Subject:
plant viruses, peanuts, necrosis, Groundnut bud necrosis tospovirus, genes, Thrips palmi, absorbance, polyclonal antibodies, nucleocapsid, clones, enzyme-linked immunosorbent assay, denaturation, transmission electron microscopy, viruses, Pergularia daemia, Arachis hypogaea, leaves, coat proteins, complementary DNA, polymerase chain reaction, nucleic acid annealing, inoculum, epidemiology, surveys, weed hosts, nucleotide sequences, RNA, crops, India, Lithuania
Abstract:
Orthotospoviruses (Tospoviridae) are exclusively thrip (Thysanoptera) transmitted plant viruses that affect wide varieties of crops worldwide. Among them, groundnut (peanut) bud necrosis virus (GBNV) is a serious virus of groundnut (Arachis hypogaea) and is transmitted by Thrips palmi in a persistent manner (Daimei et al. 2017). In the epidemiology of orthotospoviruses, weed hosts play a vital role as a source of inoculum. In this regard, during a survey in October 2017, we observed chlorotic ring spots on the leaves of the weed Pergularia daemia (Forsk) Chiv (Asclepiadaceae), which was growing in the hedges surrounding groundnut fields in the Salem district of Tamil Nadu, India (11.625531°N, 78.006284°E) with a disease incidence of 22% (total 200 plants). Quasi-spherical-shaped particles of 80 to 90 nm were visualized in transmission electron microscopy (Hitachi HT7700, Japan) of dip preparations. Furthermore, in a direct antigen-coating enzyme-linked immunosorbent assay using GBNV polyclonal antibodies (IIHR, Bengaluru, India), all five samples assayed tested positive. The mean absorbance of the infected samples was 0.921 compared with 0.295 in the negative controls. In addition, molecular confirmation was carried out by extracting total RNA from the leaves of five symptomatic as well as asymptomatic plants using an ISOLATE II RNA Mini Kit (Bioline, London, UK), and cDNA was synthesized using a RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Lithuania) according to the manufacturer’s instructions. The cDNA was subjected to polymerase chain reaction (PCR) using newly designed primers specific to the nucleocapsid gene of GBNV (forward, 5′-ATCGATCATATGTCTAACGTCAAGCAACTC-3′; and reverse, 5′-CTAGGGATCCTTACAATTCCAGCGAAGGACC-3′). PCR conditions were as follows: initial denaturation at 94°C for 3 min, followed by 35 cycles of denaturation at 94°C for 45 s, annealing at 55°C for 1 min, and extension at 72°C for 90 s, followed by a final extension at 72°C for 20 min. The PCR assay yielded the expected amplicon size of 830 bp only in the symptomatic samples. For sequencing, the amplicons of two samples were cloned into the pTZ57R/T vector (Thermo Scientific), and three clones of each sample in both directions were sequenced using a universal M13 primer. The sequences were 100% identical. BLAST analysis confirmed the presence of GBNV in P. daemia, and the sequence was deposited in GenBank (MG759525). Pairwise comparison of the sequences with corresponding nucleotide sequences of GBNV showed a 97 to 98% identity in the coat protein region with other GBNV sequences (AY618567, AY882003, EU373768, and AY882004) from India. These results confirmed the presence of GBNV in P. daemia. To the best of our knowledge, this is the first documented serological and molecular evidence for the infection of GBNV in P. daemia in India.
Agid:
6219207