L. Tomassoli

Oligonucleotide microarray-based detection and identification of 10 major tomato viruses.

A DNA microarray chip was developed for screening 10 major economically important tomato viruses from infected plants using Combimatrix platform 40-mer oligonucleotide probes. A total of 279 oligonucleotide virus probes were specific for simultaneous multiple detection, identification, differentiation and/or genotyping of each of the following tomato RNA viruses and/or strains and a virus satellite: Cucumber mosaic virus, Cucumber mosaic virus satellite RNA, Tomatoinfectiouschlorosisvirus, Tomato chlorosisvirus, Tomato spotted wilt virus, Pepino mosaic virus, Potato virus Y, Tobacco mosaic virus and Tomato mosaic virus. This selection included both positive and negative single-stranded RNA viruses. The single-stranded DNA viruses, Tomato yellow leaf curl virus and Tomato yellow leaf curl Sardinia virus were detected but were not differentiated using probes designed from their coat protein genes. A sectored oligonucleotide microarray chip containing four sets of 2000 features (4 x 2 K) was designed. In this way, four samples were tested simultaneously in a hybridization event and 16 samples were analyzed by re-using the chip four times. The hybrids had low background signals. Many of the 40-mer oligonucleotide probes were specific for the detection and identification of each RNA viral species, RNA viral satellite and genotyping strains of Cucumber mosaic virus, Pepino mosaic virus and Potato virus Y. Universal probes were developed for strains of the last three viruses and also for the genus Tobamovirus which includes both Tobacco mosaicvirus and Tomato mosaic virus.

IRIS YELLOW SPOT VIRUS ON ONION CROPS IN CALABRIA, SOUTHERN ITALY

The red onion (Allium cepa) cv. Cipolla rossa di Tropea, is a crop of utmost relevance in Calabria (southern Italy) as it consti- tutes the fourth most important agricultural product of Italy en- dowed with a certified protected geographical indication. In May 2012, symptoms resembling those of Iris yellow spot virus (IYSV), consisting of diamond-shaped lesions on the scapes ac- companied by chlorotic or necrotic spots on the leaves, were ob- served in several commercial plots. Symptomatic plants were col- lected from bulb and seed crops and tested for the presence of IYSV, a virus recorded from northern Italy since 2008 (Tomassoli et al., 2009). Total RNA was extracted from scapes and leaves and tested by single-step RT-PCR using primers specific to the vi- ral nucleocapsid (N) gene (Tomassoli et al., 2009). Five of 14 test- ed samples were IYSV-positive, two from fresh market bulb and three from seed crops. Amplicons (ca. 600 bp) were purified, se- quenced and three of the sequences were deposited in GenBank (accession Nos. JX310661, JX310662, JX310663). IYSV isolates from Calabria showed 98.5 to 100% identity at the amino acid level with the N gene sequences of the isolates previously identi- fied in Italy (FJ185142), Serbia and Brazil (EU750697 and AF067070). Real time RT-PCR assays disclosed a higher number (10) of infected samples, confirming the highly sensitive and rap- id detection of IYSV in onion afforded by this technique (Tiberi- ni et al., 2011). To our knowledge, this is the first report of IYSV in Calabria. It confirms spreading of the virus within Italy and calls for surveillance for reducing the impact of IYSV infections on onion crops and other allium species.