Detection and characterization of Tomato spotted wilt virus infecting field and greenhouse-grown crops in Zimbabwe

Eur J Plant Pathol DOI 10.1007/s10658-017-1243-4 Detection and characterization of Tomato spotted wilt virus infecting field and greenhouse-grown crops in Zimbabwe Charles Karavina & Augustine Gubba Accepted: 3 May 2017 # The Author(s) 2017. This article is an open access publication Abstract Tomato spotted wilt virus (TSWV), the type species of the genus Tospovirus in the family Bunyaviridae, is one of the most economically important emerging plant viruses worldwide. It causes over US$1 billion losses annually in open field and greenhouse-grown crops. A study was carried out to determine the geographical distribution, host range and phylogeny of TSWV in Zimbabwe. Disease surveys were conducted in 18 districts over a three-year period using tospovirus immunostrips. Virus-infected leaf samples were collected on FTA cards and in RNAlater solution. TSWV was characterized by double antibody sandwich-enzyme linked immunosorbent assay (DASELISA) and reverse transcription polymerase chain reaction (RT-PCR) followed by sequencing and phylogenetic analysis. The virus was detected in 50% of the districts surveyed, mostly in the country’s prime agricultural region. It was confirmed to be present by DASELISA and RT-PCR. In addition to some previously reported hosts, TSWV was detected for the first time in Cucurbita moschata, Cucurbita pepo, Cucumis Electronic supplementary material The online version of this article (doi:10.1007/s10658-017-1243-4) contains supplementary material, which is available to authorized users. C. Karavina : A. Gubba Department of Plant Pathology, University of KwaZulu-Natal, Private Bag X01, Pietermaritzburg, South Africa C. Karavina (*) Department of Crop Science, Bindura University of Science Education, Private Bag 1020, Bindura, Zimbabwe e-mail: sativus and Gyposphila elegans. Molecular analysis of the TSWV nucleocapsid gene showed that Zimbabwean TSWV isolates were highly similar (≥93.99%) and identical (99.02%) to each other at nucleotide and amino acid sequence levels. When compared to isolates from the rest of the world, Zimbabwean TSWV isolates were most closely related to isolates from Italy, Montenegro, New Zealand and Serbia. The study lays the foundation for future TSWV studies by providing protocols and procedures that could be used. Information generated in this study will be useful in formulating effective TSWV control measures. Keywords Immunostrip . Host . Phylogenetic analysis . RT-PCR . Tospovirus Introduction Tomato spotted wilt virus (TSWV), the type species of the genus Tospovirus in the family Bunyaviridae, is one of the most economically important emerging plantinfecting viruses worldwide (Tsompana et al. 2005; Pappu et al. 2009; Scholthof et al. 2011). It causes losses estimated at US$1 billion annually for several important ornamental, food and cash crops worldwide (Goldbach and Peters 1994; Parrella et al. 2003). It infects over 1300 species in more than 92 plant families (Parrella et al. 2003; EFSA 2012). Since its first report in Australia in 1915 (Brittlebank 1919), TSWV has been reported in over 60 countries worldwide. Eur J Plant Pathol TSWV has a tripartite single-stranded RNA genome consisting of the large (L), medium (M) and small (S) RNA segments. The L RNA encodes the RNAdependent RNA polymerase in the negative sense. The M RNA encodes two proteins: the non-structural movement protein (NSm) in the viral sense and the Gn-Gc glycoprotein in the nonviral sense. The S RNA encodes the non-structural protein (NSs) in the viral sense and the nucleocapsid (N) protein in the nonviral sense (Whitfield et al. 2005; Pappu et al. 2009). The TSWV virion is quasi-spherical, 80–120 nm diameter, and enveloped by a double membrane (German et al. 1992). Currently, at least nine thrips (Thysanoptera: Thripidae) species are known to transmit TSWV in a persistent-propagative manner, with Frankliniella occidentalis Pergande being the primary vector (Riley et al. 2011; Rotenberg et al. 2015). The virus induces a wide range of symptoms in its hosts depending on environmental factors, host cultivar, pathogen strain and stage of host development (Sevik and Arli-Sokmen 2012). Common symptoms include ringspots, line patterns, mottling and chlorotic blotches on leaves, severe stunting, wilting, and even plant death. TSWV-infected young tomato (Solanum lycopersicum L.) plants are characterized by inward cupping of leaves, bronzing and deformed fruits which show uneven ripening and raised bumps on the surface (Sether and DeAngelis 1992). In pepper (Capsicum annuum L.), severe stunting and chlorotic or mosaic yellow flecking of leaves are observed in infected plants. Necrotic spots are also present on pepper fruits, which often display ring patterns (Turina et al. 2012). In Zimbabwe, TSWV was first reported infecting tobacco (Nicotiana tabacum L.) in 1940 (Hopkins 1940). It was subsequently reported infecting at least 26 other vegetable, ornamental and weed species (Masuka et al. 1998; Nyamupingidza and Machakaire 2003). Recent studies have pointed out that the worldwide TSWV host range is expanding (Pappu et al. 2009), in part due to agricultural diversification and intensification, climate change, mutations and virus introductions into new habitats due to global trade in agricultural commodities (Hoffmann et al. 2001; Sharman and Persley 2006). New hosts are being reported mainly in tropical and sub-tropical environments where the disease is now prevalent (Robbins et al. 2010; Macharia et al. 2016). This necessitates the study of TSWV host range in a sub-tropical country like Zimbabwe. Moreover, Zimbabwean TSWV records are outdated, with no new records since 1998 (Masuka et al. 1998). The available records do not indicate how TSWV detection and characterization were done. In the last three decades, advances in the discipline of virology have revolutionized plant virus diagnostics. For example, modern virological studies incorporating molecular assays followed by phylogenetic analyses have been employed to help avoid ambiguity in tospoviral identification (Okuda and Hanada 2001; Sivparsad and Gubba 2008). Correct TSWV identification is crucial for effective disease management. In this study, we investigated the occurrence of TSWV in Zimbabwe. Specifically, we assessed the geographical distribution and host range, outlined protocols for the detection and characterization of Zimbabwean TSWV isolates, and assessed the phylogenetic relationship of Zimbabwean TSWV isolates to those isolates that occur worldwide. Materials and methods Sampling and field detection of tospoviruses TSWV disease surveys were conducted from December 2013 to October 2015 in 18 districts across six provinces of Zimbabwe (Fig. 1). For Harare Province, the three surveyed districts were Harare East, Harare West and Harare North. The surveys targeted agricultural research institutions, irrigation schemes, plots, nutrition gardens, and commercial and communal farms where a (...truncated)