Antonio Tiberini

Optimization and improvement of oligonucleotide microarray-based detection of tomato viruses and pospiviroids

Tomato (Solanum lycopersicum L.) is a vegetable crop which is affected by many viruses and several viroids, causing significant economic loss. Their detection and identification is of critical importance for plant protection and quarantine and certification programs. The potential was examined of an array based on the Combimatrix platform for the detection of 37 viruses belonging to 13 families, one of which is unassigned, together with six pospiviroid species, genus Pospiviroid, family Pospiviroidae. More than 470 oligonucleotide probes (40-mer) were selected for the microarray diagnostic technique developed in this investigation. Most of the virus probes were highly specific and were able to identify tomato viruses. Most pospiviroid probes, however, were non-specific in terms of species, but were specific at the genus level as they hybridized to members of the genus Pospiviroid. Only one probe of the Tomato apical stem viroid was species specific. The repeatability and specificity of the Combimatrix method showed that it can be considered for routine diagnostic use in suspected tomato germplasm since it detected 37 viruses and one pospiviroid at the species level and 5-6 pospiviroids at the genus level. The estimated cost for testing of a single tomato virus is similar to or less than the cost of using ELISA.

Viruses of asparagus.

The current knowledge on viruses infecting asparagus (Asparagus officinalis) is reviewed. Over half a century, nine virus species belonging to the genera Ilarvirus, Cucumovirus, Nepovirus, Tobamovirus, Potexvirus, and Potyvirus have been found in this crop. The potyvirus Asparagus virus 1 (AV1) and the ilarvirus Asparagus virus 2 (AV2) are widespread and negatively affect the economic life of asparagus crops reducing yield and increasing the susceptibility to biotic and abiotic stress. The main properties and epidemiology of AV1 and AV2 as well as diagnostic techniques for their detection and identification are described. Minor viruses and control are briefly outlined.

DEVELOPMENT OF A OLIGONUCLEOTIDE-BASED MICROARRAY FOR DETECTION OF MULTIPLE ARTICHOKE VIRUSES

Globe artichoke (Cynara scolymus) is a vegetable crop affected by many viruses causing significant economic losses. Virus detection and identification is important to farmers, plant pathologists and others involved in plant protection activities of quarantine and certification programs. To this aim, the potentiality of an oligonucleotide diagnostic array based on the Combimatrix platform for the detection and identification of 14 artichoke-infecting viruses was examined. More than 190 oligonucleotide virus probes (40-mer) were selected and used in the microarray diagnostic technique developed in this investigation. Most of the probes were highly specific for detection, identification and discrimination of target viruses. Further, for the first time the Combimatrix platform was applied directly to evaluate the sanitary status of artichoke samples collected during field surveys carried out in 6 different Italian regions. The repeatability, specificity and sensitivity of the oligonucleotide diagnostic array developed in this investigation demonstrated its applicability for routine diagnostic use in artichoke germplasm as it detected simultaneously 14 viruses in one single hybridization event.

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.

Incidence and genetic variability of Asparagus virus 1 in naturally infected asparagus.

A study to determine the incidence of viral diseases in green asparagus ( Asparagus officinalis L.) was carried out in Italy, sampling spears, young stems and ferns from com- mercial fields in several regions. Asparagus bunches from supermarkets originating from Italy as well as from sev- eral other countries, were also examined. Molecular assays (RT-PCR) were developed for the simultaneous detection of Asparagus virus 1 (AV-1) and Asparagus virus 2 (AV-2). The incidence of the two viruses was quite different. AV-2 was restricted to a few Italian cultivation areas and was never detected in samples from Spain, Peru, Mexico, Ec- uador, Argentina, or the USA. In contrast, AV-1 was widely spread in Italian asparagus crops with an average infection rate of about 50.7% and was detected in imported aspara- gus at different rates. Partial sequences of AV-1 isolates from different geographical areas were obtained. Their 3’ terminal genomic region comprising the partial coat protein gene and the 3’ untranslated region of 18 isolates were compared to each other and to species of the genus Potyvirus . Results revealed that AV-1 has become the most widespread virus in commercial asparagus crops of Italy, and presumably in other asparagus producing countries. Further, our study provides, for the first time, valuable information on the molecular variability of AV-1 isolates from different parts of the world.

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.