First Report of Broad bean wilt virus 2 and Ranunculus mild mosaic virus in Ranunculus asiaticus in Southern Italy

During 2013 to 2014, about 20% of Ranunculus asiaticus cv. Elegance plants in a commercial greenhouse in Campania, Southern Italy, were found showing leaf deformation and mosaic. Leaves from twenty-five symptomatic plants were tested by double-antibody sandwich (DAS)-ELISA using commercial diagnostic kits (Loewe, Munich, Germany) against Broad bean wilt virus 1 (BBWV-1), Broad bean wilt virus 2 (BBWV-2), Cucumber mosaic virus (CMV), Impatiens necrotic spot virus (INSV), Potato virus Y (PVY), Turnip mosaic virus (TuMV), and Tomato spotted wilt virus (TSWV). Commercial positive and negative controls (Loewe) were included in each ELISA. The same samples were also analyzed using fast lateral flow kits provided by Istituto per la Protezione Sostenibile delle Piante (IPSP), Torino, Italy, using polyclonal antibodies against Ranunculus leaf distortion virus (RanLDV-RN122), Ranunculus mild mosaic virus (RanMMV-RN129), and Ranunculus mosaic virus (RanMV-RN136) (Turina et al. 2006). All twenty-five symptomatic buttercup plants tested positive for BBWV-2, genus Fabavirus, family Secoviridae; twelve of them also tested positive for RanMMV, genus Potyvirus, family Potyviridae, a virus previously reported on this species in Italy (Turina et al. 2006). Electron microscopy of symptomatic infected leaves confirmed the presence of isometric and filamentous particles of about 25 and 800 nm, respectively, corresponding in size and shape to BBWV-2 and to RanMMV. Sap from the symptomatic buttercups infected with BBWV-2, when inoculated onto indicators, induced the following symptoms: chlorotic and necrotic lesions on inoculated leaves, systemic chlorotic mottle, epinasty, and apical necrosis on upper leaves on Chenopodium amaranthicolor, C. murale, and C. quinoa; local and systemic chlorotic ringspots on Nicotiana glutinosa and N. tabacum cv. White Burley; local and systemic necrotic spots, mild mosaic on N. occidentalis; mosaic on N. bentamiana and N. rustica. No symptoms were observed on Datura stramonium, Solanum esculentum, and Ocimum basilicum. RanMMV was not mechanically transmitted to indicators. BBWV-2 was successfully mechanically transmitted from C. amaranthicolor via three single-lesion passages to five buttercup plants and subsequently showed symptoms identical to those observed in the original symptomatic plants from the commercial greenhouse. Transmission data were confirmed by serological analysis. To confirm BBWV-2 and RanMMV infection, total RNAs (Dalmay et al. 1993) extracted from leaves of three symptomatic buttercups were reverse transcribed to cDNA using random primers and M4T (Chen et al. 2001), respectively. Primer pairs Fab5R1F/Fab5R1R (for specific detection of Fabavirus genus; Li et al. 2012) and Sprimer/M4 (for specific detection of Potyvirus genus; Chen et al. 2001) were used for PCR analysis. Amplicons of the expected size of 390 bp (BBWV-2) and 1,800 bp (RanMMV) were obtained from infected buttercup samples and directly sequenced. Using BLASTn, the Ranunculus BBWV-2 isolate sequence (GenBank Accession No. KJ715961) showed 94 to 97% nucleotide identity with more than 28 BBWV-2 isolates in GenBank, including a Korean isolate from pepper (JX183225) and an isolate from tomato in Xinjiang (FN985164). The RanMMV isolate sequence (KJ715960) showed 97 to 98% nucleotide identity with the three other GenBank isolates from Italy, Israel, and China (DQ152191, EF445546, and EU684747, respectively). To our knowledge, this is the first report of R. asiaticus as a natural host of BBWV-2 and the first report of RanMMV infecting this species in Campania, Southern Italy. © 2016, American Phytopathological Society. All rights reserved.