First Report of a New Curtovirus Species, Spinach severe curly top virus, in Commercial Spinach Plants (Spinacia oleracea) from South-Central Arizona.

Abstract

During April 2009, a commercial spinach field (1 km2 [250 acres]) in south-central Arizona developed geminivirus-like disease symptoms (4). Approximately 40 to 50% of the spinach plants exhibited extreme leaf distortion, foliar interveinal chlorosis, shortened internodes, and ~80% yield reduction. The beet leafhopper, Circulifer tenellus, the only known insect vector of curtoviruses in the United States, was observed on spinach plants. Total DNA was isolated (1) from three plant samples exhibiting the same symptom phenotype and used to PCR-amplify a 446-bp fragment of a suspected curtovirus, using primers F 5'-CTACCATCAGTAATGATGGG-3'and R 5' CATATTTGCCACCTCCAGTGTC-3' designed around the coat protein gene (Cp) for several known curtoviruses. DNA sequencing and BLAST analysis of the cloned fragments (n = 3 with 100% identity) revealed BLAST matches at 81 to 83% with the Cp for three isolates of Beet curly top Iran virus (BCTIV) (EU273816-18). To amplify the full-length curtovirus genome, total DNA from one of the three positive samples was used as the template in rolling circle amplification (RCA) employing the non-sequence specific TempliPhi 100 Amplification System (GE Healthcare) that amplifies circular DNA templates. The RCA products were linearized with PstI, yielding a ~3-Kbp fragment that was cloned into pGEM3zf+ (Promega, Madison, WI). To obtain the complete sequence, one plasmid (09-10-8) containing a full-length insert was selected and prepared for sequencing with the Template Generation System II Kit (Finnzymes, Espoo, Finland). The resultant 28 sequences were assembled into a contig using SeqMan software (DNASTAR, Madison, WI). Also, RCA clones (09.10-2, -3, and -4) from the same sample were subjected to DNA sequencing with universal M13F and M13R primers followed by primer walking (>300 bp overlap). The four 3,066-bp genomes shared 99 to 100% nt identity. An alignment (ClustalV; MegAlign, DNASTAR) with sequences of all curtovirus species available in GenBank indicated that the Arizona spinach isolates shared the highest nt sequence identity (59%) with Horseradish curly top virus (HrCTV). The next closest relatives were Beet mild curly top virus, Beet severe curly top virus, and Spinach curly top virus, at 50%. The genome consists of six open reading frames and lacks the AC3 gene, an arrangement most similar to HrCTV (3). The ICTV approved working cut-off for Curtovirus species demarcation at <89% nt identity (2) supports recognition of this isolate from spinach (GU734126) as a new, previously undescribed curtovirus species, for which we propose the name Spinach severe curly top virus (SSCTV-[Arizona:2009]). The curtovirus-like symptoms, presence of the curtovirus leafhopper vector, and isolation of a curtovirus-like genome from symptomatic spinach plants are highly suggestive of curtovirus etiology. To our knowledge, this is the first report of SSCTV worldwide and its association with diseased spinach in Arizona. Although a different curtovirus species was reported from the same infected spinach field (4), this study provides evidence that at least two curtoviruses were present in this spinach field in Arizona.