In July 2014, a sample of Pittosporum tobira (cultivar unknown) from a landscape in Isle of Wight County, Virginia, with symptoms of foliar chlorotic ring spots and line patterns, was submitted to the Virginia Tech Plant Disease Clinic for diagnosis. The sample tested positive for Tomato spotted wilt virus (TSWV) (family Bunyaviridae, genus Tospovirus), but negative for Impatiens necrotic spot virus with immunostrips from Agdia, Inc. (Elkhart, IN). To confirm the diagnosis of TSWV and rule out Tomato chlorotic spot virus (TCSV) and Groundnut ringspot virus (GRSV), with which the immunostrips are known to react, we extracted total RNA from the symptomatic leaves and performed reverse-transcription PCR and comparative sequence analysis. An ∼800-bp cDNA fragment (GenBank Accession No. KT452081), amplified with a primer pair specific for TSWV L RNA (Martínez et al. 2014), was cloned and sequenced, and determined to have 98% nucleotide identity with the corresponding sequence from several TSWV isolates, including Accession Nos. KJ575619.1, KC261971.1, and KC767959.1. Next we amplified, cloned, and sequenced the full-length cDNA of TSWV S RNA (3014 bp, KT452079) with the primer pair, S1F and S3R (Lee et al. 2011). This cDNA sequence was found to have 99% nucleotide identity to TSWV isolate AY744477.1, and 98% identity to TSWV isolates AF020659.1 and AY870392.1. A 2.1-kb fragment of the TSWV M RNA was amplified with the primer pair M1F and M2R (Lee et al. 2011). A sequence of 978 nucleotides (nt 40 to nt 986, which includes the NSm coding sequence, KT452080) had 97% identity to the corresponding sequence of five TSWV isolates, AY744487.1, AY744489.1 to AY744491.1, and AY870390.1, but only 79.5% identity to GRSV (AF213673.1) and 80.3% identity to TCSV (AF213674.1). We also used a primer pair, J060 and J064, designed to amplify S RNA of all three viruses (Hassani-Mehraban et al. 2010). The single PCR fragment of 714 nucleotides had 99% identity to the S RNA N gene of multiple TSWV isolates from North Carolina and California (AY744468.1 to AY744474.1). Sequence identities with GRSV and TCSV were 76.7% and 75.7%, respectively. The high similarities among the TSWV isolate from P. tobira and multiple TSWV isolates confirmed the diagnosis of TSWV and the absence of TCSV and GRSV in the P. tobira plant with symptoms typical of tospovirus infection. We designate this isolate as TSWV-Pt-VA. Although TSWV has been reported in numerous herbaceous crops and weeds in the United States and worldwide, the only previous report of this virus in P. tobira is from Israel (Gera et al. 2000). The symptomatic Virginia P. tobira plant had been in the landscape for at least 16 years before developing symptoms; therefore, it is unlikely that the virus was present in the plant when purchased. Based on a BLAST search, the N gene sequence of TSWV-Pt-VA has 99% identity to numerous peanut TSWV isolates from an adjacent county in Virginia, suggesting thrips transmission from a regional peanut or weed host. To our knowledge, this is the first report of TSWV on any woody species other than Hydrangea macrophylla in the United States, and represents an expansion of the host range of this commercially important virus. © 2016 The American Phytopathological Society.
