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April 2016, Volume 100, Number 4 Page 870 http://dx.doi.org/10.1094/PDIS09151021PDN
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First Report of Tomato spotted wilt virus Infecting Butternut Squash (Cucurbita moschata) in Zimbabwe
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C. Karavina, J. D. Ibaba, and A. Gubba, Discipline of Plant Pathology, School of Agricultural, Earth and Environmental Sciences, University of KwaZuluNatal, Scottsville 3209, Pietermaritzburg, South Africa.
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Article History Print: 23 Mar 2016 Ahead of Print: 4 Mar 2016 First Look: 27 Oct 2015 Accepted: 19 Oct 2015
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ABSTRACT
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Butternut squash (Cucurbita moschata Duch.) is widely grown in Zimbabwe as a food and cash crop. It is prone to attack by many biotic agents resulting in significant crop yield loss. During the 2014–15 summer season, butternut squash plants growing at a Harare farm displayed symptoms of viral etiology, characterized by leaf mottling, crinkling, and mosaics. Disease incidence was estimated at 30%. Five symptomatic leaves tested on the spot, using LoeweFast Lateral Flow Kits (LOEWE Biochemica GmbH, Sauerlach, Germany) specific to three tospoviruses: Tomato spotted wilt virus (TSWV), Tomato chlorotic spot virus (TCSV), and Groundnut ringspot virus (GRSV), were all positive. Sap from infected leaves was then expressed onto FTA Whatman Elute Cards and dried at room temperature for 30 min. Knowing that TSWV infects cucurbits and has been reported in Zimbabwe, total nucleic acid recovered from the FTA cards was used to confirm TSWV presence using reverse transcription (RT)PCR. First strand cDNA synthesis was done using the reverse primer TSWV 723 (CACAAGGCAAAGACCTTGAG) and RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, USA) following the manufacturer’s instructions. PCR was performed using KAPA2G Fast HotStart ReadyMix (Kapa Biosystems), the forward primer TSWV 722 (GCTGGAGCTAAGTATAGCAGC), and the reverse primer TSWV 723. These primers amplify 620 bp of the nucleocapsid protein (N) gene of TSWV (Adkins and Rosskopf 2002). Amplicons of the expected size were obtained from all symptomatic samples tested. Amplicons were gel purified using QIAquick Gel Extraction Kit (Qiagen, Germany) before being cloned into the pCR2.1 vector of TOPOTA Cloning Kit (Life Technologies, USA). Three positive clones were sequenced at Inqaba Biotechnical Laboratories (Pretoria, South Africa) using M13 forward and reverse primers. The BLAST result of the consensus sequence generated (Accession No. KT732271) revealed that the N gene portion of the Zimbabwean TSWV infecting butternut squash shares the highest nucleotide identity of 99% with several TSWV isolates including KM379142 (Capsicuminfecting from Turkey), JN601521 (lettuceinfecting from Jordan), KF184652 (Tomatoinfecting from Serbia), KC1825566 (Capsicuminfecting from Serbia), KC4944501 (tomatoinfecting from New Zealand), and JQ692106 (chrysanthemuminfecting from Serbia). To our knowledge, this is the first report of TSWV infecting butternut squash in Zimbabwe. Given the importance of butternut squash in Zimbabwe, the occurrence of TSWV may negatively affect crop yield, farmers’ income and household food security.
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Adkins, S., and Rosskopf, E. N. 2002. Plant Dis. 86:1310.
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