Evidence of new viruses infecting Freesia hybrids showing necrotic disease

Necrotic disorder of freesia is a high impact disease for this attractive ornamental crop and is now widespread in temperate world regions, being reported and studied in Europe, United States, New Zealand and South Korea. The presence of Freesia sneak virus (FreSV, genus Ophiovirus, family Ophioviridae) has been widely associated with the necrotic disease but other viral agents might be involved in producing the heavy necrotic symptomatology of this vegetatively propagated bulbous floral crop, so severely threatened by the disorder. During the 2014 growing season, freesia plants of different cultivars with heavy necrotic symptoms were collected in the Sanremo area, Northern Italy. Symptomatic foliar tissue was used both for mechanical inoculation to the model plant Nicotiana benthamiana and for virus purification using a method applicable to multiple viruses. A total RNA extraction of the sample enriched in viral agents was tested by Sequence-Independent Amplification to get preliminary information about the presence of both known and previously undescribed viruses. In parallel, Next Generation Sequencing (NGS) of total RNA was performed on samples extracted from the inoculated N. benthamiana plants, to more easily identify unknown virus(es), due to the fact that, unlike the freesia genome, the genome of N. benthamiana is available. A total of almost 25 million reads were obtained, then assembled into about 33 thousand contigs. NGS data analysis is currently under elaboration and validation but preliminary results show possible occurrence of previously undescribed DNA and RNA viruses infecting diseased freesia. PCR tests are being implemented to validate detection of such novel virus sequences.