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IBBR publication #1733

First report of Colombian datura virus in Mandragora autumnalis in Sicily, Italy

Pacifico D, Crucitti D, Stigliano E, Ciuffo M, Vallino M, Carimi F

Plant disease 100 (11): 2338-2339. (2016)
doi: 10.1094/PDIS-04-16-0445-PDN

Colombian datura virus (CDV) is a member of the family Potyviridae transmitted by aphids in a nonpersistent manner, by mechanical inoculation and grafting. CDV was initially isolated from ornamental Brugmansia spp. from Colombia (Kahn and Bartels 1968). Since then, this virus has been detected in different solanaceous species in Europe (Schubert et al. 2006). In Italy, CDV was detected for the first time in Brugmansia spp. from private gardens and one nursery in the Apulia region (Vovlas et al. 2009). In September 2014, flower and leaf mottle symptoms were observed on autumn mandrake plants, Mandragora autumnalis (Bertol., 1820; family Solanaceae), growing in a natural reserve of the northwest Sicilian coast (Capo Gallo, PA). Considering the presence of several mandrakes showing the same symptoms, a putative virus infection was assumed although no aphids or other insect vectors were found on symptomatic plants. Filamentous particles, typical of a putative member of the family Potyviridae, were observed by transmission electron microscopy in leaf dip preparations negatively stained with 0.5% uranyl acetate. To isolate the causal agent, the sap from infected mandrakes was mechanically inoculated to Nicotiana tabacum cv. White Burley and N. benthamiana maintained in insect proof greenhouse. Chlorotic and mottled symptoms appeared on tobacco and N. benthamiana 1 week after the inoculation, while leaf wilting and tip collapse appeared progressively only on N. benthamiana, leading to plant death within 4 weeks. RNA was extracted from the mandrake and tobacco leaves with the E.Z.N.A Plant RNA Kit (Omega Biotek, Norcross, GA) and used as a template to confirm potyvirus infection. Reverse transcription was carried out with the M-MLV reverse transcription (Invitrogen, Carlsbad, CA) using random hexamers according to the manufacturer’s instructions, while the following PCR step was carried out with potyvirus-universal primers (van der Vlugt et al. 1999), using the Platinum Taq polymerase (Invitrogen). PCR products of expected size were amplified from two mandrake and one tobacco isolates, purified and cloned in the pGEM-T easy vector (Promega, Madison, WI). Sequences were determined from at least three independent clones of each isolate showing among them 100% identity at nucleotide level. A 675-bp consensus sequence was deposited in GenBank with accession no. KU159725. According to BLASTn and BLASTp analyses, this fragment showed 99 and 100% identity to nucleotide and amino-acid sequences of European CDV isolates available in GenBank. Furthermore, CDV infection was confirmed using CDV-specific primers (Chellemi et al. 2011) through RT-PCR and sequencing of PCR products (511 bp) obtained from 9/9 symptomatic M. autumnalis. No PCR products were obtained from five asymptomatic mandrakes with both universal and CDV-specific primers. To our knowledge, this is the first report of CDV in a natural Mediterranean environment, and the first report of a potyvirus infection in mandrake. Due to its broader distribution, this perennial herbaceous species could act as a possible virus reservoir threatening the local production of solanaceous crops.

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