First Report of Tomato leaf curl New Delhi virus on Calotropis procera, a Weed as Potential Reservoir Begomovirus Host in Pakistan

Abstract

HomePlant DiseaseVol. 101, No. 6First Report of Tomato leaf curl New Delhi virus on Calotropis procera, a Weed as Potential Reservoir Begomovirus Host in Pakistan Previous DISEASE NOTES OPENOpen Access licenseFirst Report of Tomato leaf curl New Delhi virus on Calotropis procera, a Weed as Potential Reservoir Begomovirus Host in PakistanS. S. Zaidi, S. Shakir, H. J. Malik, M. Farooq, I. Amin, and S. MansoorS. S. Zaidihttp://orcid.org/0000-0002-2976-4624Search for more papers by this author, S. ShakirSearch for more papers by this author, H. J. MalikSearch for more papers by this author, M. FarooqSearch for more papers by this author, I. AminSearch for more papers by this author, and S. MansoorSearch for more papers by this authorAffiliationsAuthors and Affiliations S. S. Zaidi , Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan S. Shakir , Virology Laboratory, Center for Agricultural Biochemistry and Biotechnology (CABB), University of Agriculture, Faisalabad, Pakistan, and Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan H. J. Malik M. Farooq I. Amin S. Mansoor , Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan. Published Online:19 Apr 2017https://doi.org/10.1094/PDIS-10-16-1539-PDNAboutSections ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat In 2015–16, during field surveys of Sindh and Punjab provinces of Pakistan to observe virus symptoms on cultivated crops and nearby weeds, symptoms of leaf yellowing and yellow mosaic were observed on leaves of Calotropis procera growing around the cultivated fields. C. procera, also known as rubber bush or aak, is a common weed in Pakistan. To detect the possible presence of begomoviruses, five symptomatic plants and one asymptomatic plant were collected from Punjab and Sindh, and total DNA was extracted. Southern blot hybridization was performed using a DIG labeled probe to the Tomato leaf curl New Delhi virus (ToLCNDV) C1 gene. A strong hybridization signal was observed in symptomatic plants, while there was no signal in asymptomatic plants collected from the same fields. To further confirm the association of ToLCNDV, rolling circle amplification (RCA) was performed to enrich for the circular DNA molecules of the begomovirus genome, using phi29 DNA polymerase (Thermo Fisher Scientific, Waltham, MA). The RCA product was purified and sequenced by the Illumina MiSeq sequencing system. The Illumina NeoPrep automation system (Illumina, San Diego, CA) was used with the Illumina TruSeq Nano DNA Library Prep Kit (NP-101-1001). After sequencing, adapters were trimmed by the MiSeq Reporter Software and sequencing data were analyzed by the CLC Genomics Workbench 7.5 (https://www.qiagenbioinformatics.com). De novo assemblies were prepared following the standard quality parameters (quality score: 0.001 and Phred score: 30). The NCBI BLASTn results of assembled de novo reads confirmed the presence of ToLCNDV DNA-A and ToLCNDV DNA-B (sequence available in the database with the accession number KX710158 and KX710159, respectively). The species demarcation of ToLCNDV was also confirmed using the Sequence Demarcation Tool (SDT) program (http://web.cbio.uct.ac.za/∼brejnev/), following the revised taxonomic criteria of begomoviruses (Brown et al. 2015). The nBLAST analysis of ToLCNDV DNA-A reported in this study indicated that it was 98% identical to the ToLCNDV DNA-A reported previously in chili from Khanewal, Pakistan (DQ116880); while the nBLAST analysis of ToLCNDV DNA-B reported in this study indicated that it was 95% identical to the ToLCNDV DNA-B reported previously in Cucurbita pepo from Rawalpindi, Pakistan (KT948073). No evidence for the presence of alphasatellites or betasatellite either in the Illumina data or by PCR using universal primers for amplification of alphasatellites or betasatellites was found. Whitefly transmitted ToLCNDV (Gilbertson et al. 2015) infects several important crops like tomato, potato, cucurbits (Zaidi et al. 2016a), and cotton (Zaidi et al. 2016b). The occurrence of ToLCNDV on C. procera indicates the increasing host range of ToLCNDV, which is serious threat for other economically important crops. C. procera has been reported as a potential host for Papaya leaf curl virus (JQ407224; formerly known as Croton yellow vein mosaic virus) and croton yellow vein mosaic betasatellite (HQ631430) in India (Prajapat et al. 2012). C. procera is a common weed and may therefore serve as an alternative virus reservoir, with an important role in the epidemiology of the disease. To our knowledge, this is the first report of ToLCNDV infecting C. procera in Pakistan.