Detection of mixed virus population in freesia plants with necrotic disease

Necrotic disorder of freesia (Freesia refracta hyb., family Iridaceae) was first described in The Netherlands before 1970. In following years, the disorder was widely reported in other European countries including Northern Italy (Vaira & Milne 2008). Very recently, the same necrotic disease was also detected in Virginia, United States (Vaira et al. 2009) and New Zealand (Pearson et al. 2009). Presence of the Ophiovirus Freesia sneak virus (FreSV) has been widely associated with the necrotic disease in Europe, the United States and New Zealand but some uncertainty remains (Brandvagt et al. 2008). The freesia leaf necrosis complex has been shown to be soilborne, transmitted by Olpidium brassicae, but other infectious agents (e.g. the Varicosavirus tentatively named Freesia leaf necrosis virus) could be naturally transmitted by the same vector, thickening the plot about the disease causal agent. In 2002 Dr. Morikawa was able to differentiate a Mild mottle mosaic- and Streaking disease in tulips: the first was linked to the Tulip mild mottle mosaic virus (TMMMV, genus Ophiovirus) infection and the second to an unknown Tulip streaking associated agent, with unstable infectivity (Morikawa 2002). He also showed that development of both diseases was suppressed by fungicidal treatments of the soil, suggesting soil-borne transmission for both agents. In 2005 the same team preliminarily described Tulip streak virus, a novel virus strictly associated with tulip streak disease. This new viral agent, morphologically resembling Tenuiviruses or supercoiled Ophioviruses, has a coat protein of c. 30kDa sharing some homology with Phlebovirus (Bunyaviridae) (Morikawa et al. 2005). Freesia plants showing necrotic disease were collected in Liguria (Northern Italy) during the 2011-2012 growing season. About 40-50% of the plants were affected by typical leaf necrosis and were assayed for FreSV infection by electron microscopy (TEM) and RT-PCR tests. FreSV was detected using FreSV-specific primers in all six samples obtained by pooling several Freesia plants from different areas of the parcel, and elongated supercoiled virus particles were detected in freesia crude sap by TEM. Freesia tissue was purified using Ophiovirus procedure and the product obtained was used for total RNA extraction and for TEM visualization. Several differently shaped virus particles were visualized in the mixture and total RNA has been used for sequence-independent amplification (SIA) for the identification of RNA virus, following a published procedure (Agindotan 2010). Results of virus detection in infected freesia tissue are reported.