G. Albanese

Evolutionary analysis of Citrus tristeza virus outbreaks in Calabria, Italy: two rapidly spreading and independent introductions of mild and severe isolates

The evolution of citrus tristeza virus (CTV) from outbreaks occurred in Calabria, Italy, was compared with that of CTV outbreaks reported previously in another two proximal Italian regions, Sicily and Apulia. Examination of four genomic regions (genes p20, p25 and p23, and one fragment of open reading frame 1) showed two recombination events, and phylogenetic analysis disclosed two divergent CTV groups in Calabria: one formed by severe and the other by mild isolates. This analysis, together with others involving population genetic parameters, revealed a low migration rate of CTV between the three Italian regions, as well as significant differences in selective pressures, epidemiology and demography, all affecting the genetic structure of CTV populations.

Onion yellow dwarf virus ∆∆Ct-based relative quantification obtained by using real-time polymerase chain reaction in ‘Rossa di Tropea’ onion

As part of a plant-pathogen interaction study between Onion yellow dwarf virus (OYDV) and onion cultivar Rossa di Tropea, a ΔΔCt-based relative quantification of OYDV was investigated to relate OYDV titer to accumulation of secondary metabolites in onion bulbs. An appropriate reference gene (RG) was required to achieve data normalization. Since no single internal control gene is universally used as an RG, multiple stably expressed reference genes were investigated. In particular, elongation factor (Elf), protein phosphatase 2A (PP2A), helicase (Hel-1), 5.8S rRNA, ubiquitin (UBQ) and ß-Actin (ß-Act) were compared one to another in both leaf and bulb tissues, at different growth and development stages, and with different infection status (healthy/OYDV-infected). Preliminary gene screening was carried out using an RT-qPCR assay (SYBR chemical), assessing both Ct values and melting curves. Expression stability of the reference genes in the sample sets was independently determined by three different software packages: geNorm, NormFinder and Bestkeeper. In contrast to Elf, PP2A, Hel-1 and ß-Act, 5.8S rRNA and UBQ proved to be the most stable RGs. An OYDV specific RT-qPCR TaqMan® assay was also developed and validated for relative quantification of OYDV titer. The assay was shown to be specific and sensitive, able to identify virus presence up to 10−6 dilution, representing a rapid and sensitive diagnostic tool for OYDV detection for application in field surveys. Finally, a ∆∆Ct method was developed, to be applied in future studies describing the molecular interaction between OYDV and onion cv. ‘Rossa di Tropea’. This approach was used to provide relative quantification of OYDV titer in samples obtained from different experimental trials.

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.