First Report of Tomato spotted wilt virus in Stevia rebaudiana in North Carolina

Tomato spotted wilt virus(TSWV) (genus Tospovirus, family Bunyaviridae) is an important plant pathogen on vegetables, ornamentals, and field crops across the southeastern United States (Abad et al. 2005). TSWV was first detected in North Carolina in 1988 in tobacco and tomato fields (Cho et al. 1995). Stevia (Stevia rebaudiana(Bertoni) Bertoni) is an emerging perennial crop in the United States, grown for 3 to 5 years with two harvests per growing season. Stevia leaves contain numerous glycosides that are extracted for use as a natural, low-calorie sweetener. In current production practices, stevia plants are started from seed in a greenhouse system and transplanted to the field 8 to 10 weeks after germination. In February 2015, virus-like symptoms were observed on stevia plants in a research greenhouse in Raleigh, NC. Symptomless plants were dug from the field in the fall of 2014 and moved to the greenhouse to make crosses. Symptoms began as mottling, distortion, and chlorosis of leaves and progressed to include necrotic leaf lesions, stem lesions, severe dieback, and plant death. Adult thrips, identified as Frankliniella occidentalis, were present throughout the greenhouse and on plant material. Leaf tissue from symptomatic plants was collected for Immunostrip testing (Agdia Inc., Elkhart, IN). All symptomatic tissue tested positive for TSWV and negative for Impatiens necrotic spot virus(INSV). Diagnosis was further confirmed through sequence analysis of a nucleocapsid (N) protein gene fragment amplified by reverse transcription polymerase chain reaction (RT-PCR). RNA was extracted from symptomatic tissue using TRIzol Reagent (ThermoFisher Scientific, Waltham, MA) and cDNA template was generated using Protoscript II Reverse transcription (New England BioLabs Inc., Ipswich, MA) and amplified using nucleocapsid primers TSWVNC5'PCI (5'-AATTACATGTCTAAGGTTAAGCTCACTAAGG -3') and TSWVNC3'XBA (5'-AATTTCTAGATTAAGCAAGTTCTGYGAGTTTTGCC-3') (Sit et al. unpublished). The amplified sequences of the RT-PCR products were 99% identical to TSWV NC-34 N-gene sequence in GenBank Accession No. (DQ777221.1). This is the first known report of TSWV infection in stevia in the United States. In 2007, Chatzivassiliou et al. observed viral symptoms in stevia in Greece and reported TSWV as the causal agent. The endemic nature of TSWV, along with the intended perennial production of stevia in North Carolina and the southeast, warrants monitoring for TSWV in greenhouse and field produc