Western Flower Thrips Can Transmit Tomato spotted wilt virus From Virus-infected Tomato Fruits

In this study, we demonstrate that western flower thrips (WFT, Frankliniella occidentalis) can acquire and transmit Tomato spotted wilt virus (TSWV) from symptomatic tomato fruits. TSWV and other thrips-transmitted tospoviruses have long been known to spread via plant propagation material such as transplants. Global dissemination of tospoviruses has also been linked, in part, to transport and trade of thrips-infested and virus-infected horticultural products. However, the role of tomato fruits transported across state and national borders has not previously been examined as a means of virus spread or as a source for thrips acquisition of virus. Tomato fruits displaying typical tospovirus symptoms were purchased from several Washington State grocery stores. Many of these symptomatic fruits tested positive for TSWV and some for Pepino mosaic virus (PepMV). First instar larvae of WFT successfully acquired TSWV from these infected tomato fruits and transmitted the virus as adults to Emilia sonchifolia plants. Symptomatic E. sonchifolia plants were confirmed positive for TSWV by lateral flow immunoassays and sequence analysis of a portion of the nucleocapsid gene. These results suggest the dissemination of TSWV (and likely other tospoviruses) and PepMV to new geographic areas by human-assisted transport of infected tomato fruits. Tospoviruses (family Bunyaviridae, genus Tospovirus) are one of the most damaging groups of plant viruses affecting a wide range of agricultural, floricultural, and horticultural crops grown in greenhouses or fields (Gilbertson et al. 2015). Unlike other vector-borne plant viruses, tospoviruses display several unique features that include an ambisense, tripartite RNA genome and circulative, propagative transmission by thrips (Thysanoptera: Thripidae) in a developmental stage-specific manner (Rotenberg et al. 2015). Besides their negative effects on crop yield (Sevik and Arli-Sokmen 2012), tospovirus infections can cause pronounced impacts on produce quality, leading to nonmarketable fruits, leaf vegetables, and ornamentals (Funderburk 2009). The striking symptoms produced by several tospoviruses on fruits include chlorotic and necrotic rings, irregular blotches, and deformation (Fig. 1). These symptomatic fruits could further reduce shelf-life in supermarkets and be cause for rejection by consumers due to poor aesthetic appeal and potentially reduced nutritional quality, resulting in financial losses to producers (Spence et al. 2006). Tomato spotted wilt virus (TSWV) is the most ubiquitous of the tospoviruses, causing major losses in multiple crops worldwide, and is recognized as one of the top 10 most economically destructive plant viruses (Scholthof et al. 2011). The worldwide distribution of TSWV and that of its principal vector-thrips species, the western flower thrips (WFT,Frankliniella occidentalis), have been attributed, in part, to their exceedingly wide host ranges, which include many cultivated crops, ornamentals, and uncultivated plant species (Parrella et al. 2003; Reitz 2009) and to increased transport of vector thripsinfested and virus-infected horticultural and floricultural products through global trade and commerce (Kirk and Terry 2003; Gilbertson et al. 2015). Consequently, preventing the invasion of both the virus and the vector into new locations has become a challenge. Globalization of agriculture has, in part, contributed to an increased risk of cross-border and cross-continental spread of pests and diseases (Hulme 2009). Regional and international trade of agricultural produce is often suspected to be one of the primary means for pathogen and pest movement. As noted in Lecoq et al. (2003), there is limited experimental evidence to support the assumption that viruses spread via vegetables and fruits, such as tomato. Some examples of viruses thought to have moved via tomato fruit include Tomato yellow leaf curl virus (TYLCV) into northern Europe (Just et al. 2014) and Pepino mosaic virus (PepMV) into and within Europe (Staniulis et al. 2012; Steffen et al. 2015). Likewise, Plum pox virus (PPV) was detected in peach fruits imported from Chile into Brazil (Rezende et al. 2016) and in intercepted fruit at an international airport in Australia (Davis et al. 2002). Reports of vector acquisition of viruses from infected fruits and transmission to noninfected plants are also rare. Lecoq et al. (2003) demonstrated that potyvirus-infected melon fruits could serve as a source for aphid transmission of Papaya ringspot virus and Zucchini yellow mosaic virus to young cucurbit plants. Similarly, aphid transmission of PPV from infected peach and apricot fruits has been demonstrated (Labonne and Quiot 2001; Gildow et al. 2004; Lowery et al. 2015). Whitefly transmission of TYLCV from infected tomato fruit to noninfected tomato plants was demonstrated by Delatte et al. (2003). Culled pepper fruits from TSWV-infected plants were implicated as a source of both virus and viruliferous thrips (Okazaki et al. 2007). Onions and onion transplants have been implicated in the spread of Iris yellow spot virus into new Corresponding author: Rayapati A. Naidu; E-mail: naidu.rayapati@wsu.edu © 2017 The American Phytopathological Society PLANT HEALTH PROGRESS ¿ 2017, Vol. 18, No. 1 ¿ Page 1 regions of the United States. (Hsu et al. 2011; Nischwitz et al. 2007; Schwartz et al. 2014). Import of fresh-market tomato fruits into the United States has grown dramatically in recent years and imported tomato fruits currently account for about one-third of tomato consumption (Zahniser et al. 2015). Consequently, alien pests and diseases carried via different types of tomato fruits, including truss tomatoes, could pose phytosanitary concerns and threats to agricultural security in the country (Gregory et al. 2006; Ferrier 2014; Perrings 2016). Since visual inspection of large volumes of imported tomatoes for pathogens, especially viruses, can be challenging and unreliable, infected fruits may escape scrutiny passing through the supply chain of international trade and inadvertently move across national or state borders (Wylie et al. 2014). Thus, international trade could be an additional avenue for longdistance dispersal of viruses via infected tomato fruits. Tomato fruits displaying symptoms consistent with tospovirus infection (Fig. 1) were observed at several grocery stores in eastern Washington State in the United States. These observations led us to test the hypothesis that infectious tospoviruses may be present in these fruits, and these fruits may contribute to the dissemination of tospoviruses by serving as a source for thrips acquisition. Virus Detection in Symptomatic Tomato Fruits During June and July of 2014, 38 tomato fruits (Roma type) displaying chlorotic spots and blotches characteristic of TSWV were purchased from 10 grocery stores in six locations in the Yakima Valley of Washington State (Table 1). In addition, three non-Roma-type tomato fruits displaying virus-like symptoms were purchased from a grocery store in New Orleans, LA (Table 1). According to the produce labels, these tomatoes were produced in Mexico, Canada, or the United States, and subsequently transported to retail stores for sale. Symptomatic slices were cut from each tomato fruit, homogenized in 0.01M phosphate buffer, pH 7.0, and tested for TSWV, Impatiens necrotic spot virus (INSV), and PepMV in lateral flow immunoassays (ImmunoStrip for TSWV STX 39300/0025, ImmunoStrip for INSV STX 20500/0025, ImmunoStrip for PepMV STX 13001/0025, Agdia, Inc., Elkhart, IN). Of the 41 fruits tested, 29 were positive for TSWV, 13 for PepMV, and none for INSV. Four tomato fruits were positive for both TSWV and PepMV. The remaining three fruits were negative for the three viruses. We did not test these three fruits for other viruses known to infect tomato plants (Hanssen et al. 2010). It has been reported recently that TSWV lateral flow immunoassays can react with other tospoviruses, such as Tomato chlorotic spot virus (TCSV) and Groundnut ring spot virus (GRSV) (Adkins et al. 2015). Therefore, additional molecular diagnostic assays were used to confirm lateral flow immunoassay results. Total nucleic acids were extracted from three tomato fruits positive for TSWV in lateral flow immunoassays using a previously described protocol (Bagewadi et al. 2015). The denatured extract was used as a template in one-step RT-PCR to amplify an approximately 620-bp fragment of the nucleocapsid (N) gene encoded by the S-RNA segment using primers TSWV723 and TSWV722 (Adkins and Rosskopf 2002). RT-PCR products were resolved in 1.5% agarose gels, stained with GelRed (Biotium, Hayward, CA), and visualized under UV light. The amplicons were gel eluted using the QIAquick Gel Extraction kit (Qiagen Inc., Valencia, CA) and cloned in pCR2.1 (Invitrogen Corp., Carlsbad, CA). Two clones per amplicon were sequenced in both orientations (Retrogen, Inc., San Diego, CA). Sequences of cDNA clones from two fruits were identical (GenBank Accession No. KY349923) and showed 99% nucleotide sequence identity with corresponding N gene sequence of TSWV isolates (GenBank Accession Nos. AY744468 to AY744473) reported from California (Tsompana et al. 2005). These results confirmed immunoassay results for the presence of TSWV in symptomatic tomato fruits. Thrips Acquisition and Transmission of TSWV From Infected Tomato Fruits WFTmust acquire the virus as larvae to transmit as adults (van de Wetering et al. 1996); therefore, for tomato fruits to serve as a source of inoculum, infectious TSWV must be present in the fruits and available to WFT larvae for acquisition. To test this hypothesis, three additional tomato fruits with typical TSWV symptoms were purchased from local grocery stores and TSWV infection was confirmed with lateral flow immunoassays as above. These fruits were then used to determine whether TSWV could be acquired by thrips larvae and transmitted to indicator plants under lab