Urtica membranacea: a new host for Tomato yellow leaf curl virus and Tomato yellow leaf curl Sardinia virus in Italy

In the autumn 2012, unusual symptoms of leaf yellowing and curling were observed on several plants of Urtica membranacea (Poiret) (Urticaceae), growing along the rows of a greenhouse tomato cultivation in southern Italy (Torre del Greco, Campania Region). Tomato plants (cv. Jama) in the greenhouse were affected by tomato yellow leaf curl disease (TYLCD) and had large population of Bemisia tabaci (Gennadius), mostly likely the Q1 and Q2 variants of the Mediterranean (MED) species (Parrella et al., 2014). Ten symptomatic U. membranacea plants were initially analyzed using the Tomato yellow leaf curl virus (TYLCV) specific lateral flow (LF) kit (Adgen Ltd, UK). The kit detected TYLCV in all symptomatic plants of U. membranacea while asymptomatic U. membranacea were found negative. To exclude possible infections of mechanically transmissible viruses, the TYLCV-infected plants were used to inoculate a range of herbaceous hosts, including species belonging to Chenopodiaceae, Solanaceae, Composite, Labiatae and Leguminosae families. During bioassays, no symptoms were observed, either local or systemic, in the inoculated plants, suggesting that symptoms observed were due to infection with geminiviruses that cause TYLCD. Since the LF kit cannot distinguish between TYLCV and Tomato yellow leaf curl Sardinia virus (TYLCSV), further analyses were conducted to check for TYLCV and TYLCSV presence, according to the protocol described in Parrella et al. (2006). Total nucleic acids (TNAs) were extracted from four different symptomatic U. membranacea plants and each sample subjected to polymerase chain reaction (PCR) with the primer pair TYCP1(+)/TYCP2(-), which direct the amplification of an ?680 bp DNA fragment from the V1 (capsid protein) gene of TYLCV and TYLCSV (Parrella et al., 2006). Three ?l of each PCR product were then digested with AvaII endonuclease in order to discriminate between TYLCV and TYLCSV (Parrella et al., 2006). Detection of recombinants between TYLCV and TYLCSV was then performed on the same TNAs, following the method described by Davino et al. (2008), using two primer pairs, designed based on the sequences of the IR regions of TYLCV and TYLCSV and that reveal the Rec type A and type B recombinants (Davino et al., 2008). Amplicons corresponding to TYLCV and TYLCSV parents and putative recombinants, were directly sequenced at Polo GGB (Perugia, Italy). Results obtained from the four plants indicated that two plants were infected by TYLCV (Acc. n. LN865156, 99.5% identical to isolate IT:T570:02, Accession number DQ317730), one by TYLCSV (Accession number LN865155, 98.3% identical to isolate TYLCSV5, Accession number EU307940) and one was infected by TYLCV and TYLCSV in mixed infection. This plant tested positive also for the Rec type A recombinant (Davino et al. 2008) (Accession number LN865157, 99.2% identical to the recombinant isolate 77-09, Accession number GU322872). Urtica membranacea is an annual herbaceous plant native to the Mediterranean region. Its natural habitat comprises uncultivated marginal areas close to disturbed areas, as well as moist and nitrogen-rich soils where the plants thrive. Therefore, it is easy to find U. membranacea as a weed of protected crops where fertigation is routinely used. This finding suggests that U. membranacea is a newly identified natural host for TYLCV, TYLCSV and a TYLCV/TYLCSV recombinant. Therefore, it may play a role in the epidemiology of TYLCD, especially in protected crops, acting as reservoir for geminiviruses responsible for TYLCD.