A New Isolate of Eclipta yellow vein virus Along with a Betasatellite Associated with Yellow Vein Leaf Curl Disease of Andrographis paniculata in India
Authors: Khan A. , Samad A. 
Published: October 2013

Geminiviruses are ssDNA viruses that infect a wide range of plant species. Andrographis paniculata (family Acanthaceae), an herb commonly known as Kalmegh, is famous for its medicinal properties such as anti-inflammatory, antibiotic, antimalarial, anticancerous, antidiabetic, antihepatotoxic, antioxidant, and is helpful in curing various diseases (1). Surveys of kalmegh fields carried out in September and October 2010 in Lucknow, India, revealed symptomatic plants that initially showed yellow veins on younger leaves and later upward leaf curling, vein clearing, chlorosis, reduced leaf size, poor inflorescence, and stunted growth leading to drastic reduction in herb yield. The disease incidence was estimated at 25 to 40%. Aphids (Myzus persicae and Aphis crassivora) failed to transmit the disease; however, similar disease symptoms developed on healthy plants after transmission by viruliferous whiteflies (Bemesia tabaci). Healthy whiteflies were used for acquisition feeding on the naturally infected twig of A. paniculata and then transferred to healthy seedlings for an overnight inoculation feeding. After inoculation feeding, whiteflies were killed by insecticide. Four out of six plants were positive after whitefly transmission. Total nucleic acids were extracted from the leaves of symptomatic and symptomless plants by modified CTAB method. PCR amplification of a 771-bp fragment of DNA, with begomovirus CP gene-specific primers (forward 5′-ATGGCGAAGCGACCAG-3′ and reverse 5′-TTAATTTGTGACCGAATCAT-3′) from symptomatic samples only, supported the presence of a begomovirus. The amplified DNA fragment was revealed in 13 out of 17 symptomatic samples. The full length DNA-A was amplified using two sets of overlapping primer pairs (F1For/F1Rev and F2For/F2Rev), generating the amplicons of ~1,200 bp and ~1,700 bp in size, respectively (3). Nine PCR positive samples were eluted from agarose gel by QIAquick gel extraction kit (Qiagen), cloned into pGEM-T Easy vector (Promega), and 16 clones were sequenced. The complete DNA-A sequence (2,739 nt) was deposited in GenBank (Accession No. KC476655). Sequence analysis showed 96% nucleotide identity with Eclipta yellow vein virus (EYVV, GQ478343) and more distant affinities (≤89%) with other begomoviruses. No DNA-B was detected in any of the samples with the universal primer pair PBL1v2040/PCRc1 (4). However, a betasatellite was identified by PCR amplification of a 1,379-bp fragment using universal primers β01 and β02 (2). Sequence analysis of this betasatellite (KC967282) associated with the present disease showed 83% to 89% identity with sequences of other betasatellites, like Ageratum yellow vein betasatellite (AJ542498), available in GenBank. There was no evidence of the presence of alphasatellites. The presence of a begomovirus and an associated betasatellite was also validated using rolling circle amplification with TempliPhi 100 Amplification system (GE Healthcare), which generated two fragments of 2.7 kb and 1.3 kb, respectively, after digestion with enzymes EcoRI, EcoRV, and HincII. EYVV (family Geminiviridae; genus Begomovirus) was reported for the first time from Pakistan in 2006 on Eclipta prostrata (GQ478343.1). To our knowledge, this is the first report of a new isolate of EYVV infecting A. paniculata in India. Kalmegh is cultivated as a mixed crop in some areas and it could potentially be a reservoir of inoculum to other hosts susceptible to begomoviruses.

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