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First Report of Tomato torrado virus on Tomato (Solanum lycopersicum) in South Africa

    Authors and Affiliations
    • V. Moodley
    • A. Gubba , Discipline of Plant Pathology, School of Agricultural, Earth and Environmental Sciences
    • P. L. Mafongoya , Rural Agronomy and Development, School of Agricultural, Earth and Environmental Sciences, University of KwaZulu-Natal, Scottsville, Pietermaritzburg, Republic of South Africa.

      During the 2015 growing season, tunnel- and field-cultivated tomato (Solanum lycopersicum L.) crops in the Limpopo Province of the Republic of South Africa developed dark brown spots on the leaves that progressed to a burnt-like appearance. Symptoms began as necrotic spots at the base of young leaves following vertical necrotic lesions on the stem. Mild chlorosis often surrounded the onset of leaf necrosis. Fruit became deformed, hardened, and blotched with necrotic tissue, making it unmarketable. These symptoms coincided with abnormally high whitefly populations in the province and are similar to those previously described from parts of Europe, South America, and Australia as a result of whitefly-transmitted torrado viruses (Verbeek et al. 2012). In Mexico, similar symptoms were reported on tomato crops infected with Tomato apex necrosis virus, a member of the Secoviridae family. Total RNA was extracted, using a Zymo Quick-RNA MiniPrep kit (Irvine, CA, USA), from symptomatic leaf tissue collected across the province from three fields and one greenhouse site. A degenerate Torradovirus primer set, Torrado-2F: TGGGATGARTGYAATGTKCT and Torrado-2R: CCWGTCCACCAYTTGCAATT (Verbeek et al. 2012), was used to amplify a 515-nt region overlapping the first two coat proteins (Vp35 and Vp26) located on RNA 2 of Tomato torrado virus (ToTV) using a two-step reverse transcription (Thermo Scientific, Waltham, MA, USA) polymerase chain reaction (KAPA 2G Universal Fast Master Mix) (RT-PCR) following the manufacturers’ instructions. Northern blot hybridization using a digoxigenin (DIG) labeled RNA probe complementary to the above-mentioned region on the RNA2 molecule was performed using the DIG Northern Starter Kit (Roche). ToTV was positively identified using RT-PCR and confirmed by means of RNA in situ hybridization in two field and one greenhouse location, on tomatoes exhibiting virus-like symptoms. In addition, the virus was detected in symptomatic jimson weed (Datura stramonium) harboring large populations of whiteflies along rows of tomato crops. A gel extraction of each positive sample was performed using a MinElute Gel Extraction kit (Qiagen, Netherlands). The purified product was cloned using a TA cloning kit (Invitrogen, CA, USA) and sequenced at Inqaba Biotec (Inqaba Biotechnical Industries, Pretoria, South Africa) using M13 primers. Analysis of the sequence data using BLAST confirmed the identity of Lim-186 (GenBank Accession No. KP890356) as ToTV. Lim-186 had a 99% nucleotide identity match to the RNA 2 segment of the isolate Wal’03 (EU563947), which was initially identified in the Wielkopolska Province of Poland by Budziszewska and colleagues in 2003 (Budziszewska et al. 2008). To our knowledge, this is the first report of ToTV in South Africa and the mainland African continent.