Dieback and wilting caused by tomato spotted wilt virus in Arctotis x hybrida in Italy

In the winter 2012 some plants of African daisy (Arctotis x hybrida L., family Asteraceae) cv. Hannah, cultivated for commercial purposes in a greenhouse located at Albenga (Savona provence, northwest Italy), were noticed for a rapid dieback, generalized reddening, following by an irreversible wilting. The percantage of affected plants was around 3-5%. African daisy leaves, arwested from three different symptomatic plants, were ground in cold (5°C ca.) phospate1 g of fresh leaves in 4 ml of sodium phosphate 0.03 M, containing 0.2% sodium diethyldithiocarbamate, 75 mg/ml of active charcoal, and traces of Carborundum (600 mesh). The inoculum was rubbed on healthy indicator host plants and inoculated plants were maintained in an insect-proof greenhouse with natural illumination and temperatures of 24 and 18°C day/night. Under these conditions, inoculated plants showed the following symptoms after 1 to 3 weeks: necrotic local lesions in Chenopodium amaranticolor and C. quinoa; yellowing and stunting following by systemic necrosis and death of the plants in tomato (Solanum lycopersicum cv. San Marzano); necrotic local lesions following by systemic necrotic patterns and leaf deformation in tobacco (Nicotiana tabacum cv. Xanthi nc.) and N. glutinosa: necrotic local lesions in petunia (Petunia x hybrida, cv. Pink Beauty). Leaf samples from the same three symptomatic plants were tested by double-antibody sandwich-ELISA with polyclonal antisera against Cucumber mosaic virus (CMV) and Tospoviruses (Tospovirus broad-spectrum, Serogroups I, II and III, Bioreba AG, Switzerland). All three plants reacted positively against Tospo-groups antibodies, but not the CMV antibodies. Total RNA was extracted from infected samples with the RNeasy Plant Mini Kit (Qiagen, Valencia, CA) and subjected to reverse transcription polymerase chain reaction (RT-PCR) by using the tospovirus universal primers BR60/BR65 which amplify part of the nucleocapsid protein gene (1). Target amplicons of 454 bp were produced for all three samples. The PCR products were cloned and sequenced on both strands (one clone per amplicon cloned). The resulting sequences were 100% identical, so a single sequence was deposited in GenBank (Accession No. XXXXX). The sequence shows highest homology (99%) to the Tomato spotted wilt virus tomato isolate NJ-JN from South Korea (Accession No. HM581936). The identity of the virus infecting African daisy was further confirmed by sequencing amplicons obtained by RT-PCR using primers coverimg partially the movement protein of TSWV. The sequence obtained (GenBank Accession No. XXXXX), shows high homology, 99%, to several isolates of the Tomato spotted wilt virus, including again the Korean tomato NJ-JN isolate (Accession Nos. AY744493, AB663306 and HM581936 ). To our knowledge, this is the first report of TSWV infecting African daisy plants and adds a new cultivated species in the Asteraceae, the family with the highest number of TSWV host species, to the list of possible reservoirs of the virus (2).