Development of three duplex real-time RT-PCR assays for the sensitive and rapid detection of a phytoplasma and five viral pathogens affecting stone fruit trees.

Three duplex real-time reverse-transcription polymerase chain reaction (real-time RT-PCR) assays based on TaqMan chemistry, were developed for the simultaneous detection and specific quantification of apple chlorotic leafspot virus (ACLSV), plum pox virus (PPV), prunus necrotic ringspot virus (PNRSV), prune dwarf virus (PDV), peach latent mosaic viroid (PLMVd) and the European stone fruit yellows (ESFY) phytoplasma, which are considered among the most important pathogens affecting stone fruit trees. The quantitative RT-PCR (RT-qPCR) assays were optimized using RNA transcripts (linearized plasmid was used for the assay optimization of the ESFY phytoplasma) of known concentrations. No differences in sensitivity were recorded between the duplex and singleplex RT-qPCR assays.

Development of a Real-Time RT-PCR for the Universal Detection of LChV1 and Study of the Seasonal Fluctuation of the Viral Titer in Sweet Cherry Cultivars.

Little cherry virus 1 (LChV1) is a sweet cherry pathogen which has lately been reported in other Prunus spp. LChV1 variability makes reliable detection a challenging undertaking. The objective of this work was to develop a rapid, sensitive, and reliable one-tube, real-time reverse-transcription polymerase chain reaction (RT-PCR) for the detection and quantification of LChV1.

Development of one-tube real-time RT-qPCR for the universal detection and quantification of Plum pox virus (PPV).

In this study a one-tube real-time RT-qPCR assay was developed using the TaqMan chemistry for the universal detection and quantification of PPV, one of the most important pathogens affecting stone fruit trees. In order to design appropriate primers and probe, nucleotide sequences from different PPV isolates originating from all known strains were recovered from the databases. Various genomic regions were screened and finally primers were selected from a conserved region of the 3'- terminal part of the CP gene amplifying a 146 bp DNA fragment while the probe was designed to bind within the amplicon.

Insights Into the Etiology of Polerovirus-Induced Pepper Yellows Disease.

The study of an emerging yellows disease of pepper crops (pepper yellows disease [PYD]) in Greece led to the identification of a polerovirus closely related to Pepper vein yellows virus (PeVYV). Recovery of its full genome sequence by next-generation sequencing of small interfering RNAs allowed its characterization as a new poleroviruses, which was provisionally named Pepper yellows virus (PeYV). Transmission experiments revealed its association with the disease.

Efficacy and feasibility of the Integrated Psychological Therapy for outpatients with schizophrenia in Greece: Final results of a RCT.

The goal of this study was to evaluate the efficacy and the feasibility of cognitive remediation group therapy in patients with schizophrenia in Greece. For this purpose, the cognitive part of the Integrated Psychological Therapy (IPT), focusing on neuro- and social cognition, was compared in a randomized controlled trial (RCT) with treatment as usual (TAU). 48 outpatients took part in the study.

A novel grapevine badnavirus is associated with the Roditis leaf discoloration disease.

Roditis leaf discoloration (RLD), a graft-transmissible disease of grapevine, was first reported in Greece in the 1980s. Even though various native grapevine viruses were identified in the affected vines, the etiology of the disease remained unknown. In the present study, we used an NGS platform for sequencing siRNAs from a twenty-year old Roditis vine showing typical RLD symptoms.

Development of one-tube real-time qRT-PCR and evaluation of RNA extraction methods for the detection of Eggplant mottled dwarf virus in different species.

A one-tube real-time qRT-PCR assay was developed, for the detection and quantification of Eggplant mottled dwarf virus (EMDV), a pathogen affecting cultivated and ornamental plants. The amplification efficiency of the assay was 98% and the linear range of quantification was from 20 to 2×10(8) RNA transcripts. Total RNA extraction methods (three developed methods and one commercially available RNA extraction kit) were evaluated using tissues from seven different plant species and synthetic EMDV RNA transcripts of known concentration.

Generic detection of poleroviruses using an RT-PCR assay targeting the RdRp coding sequence.

In this study a two-step RT-PCR assay was developed for the generic detection of poleroviruses. The RdRp coding region was selected as the primers' target, since it differs significantly from that of other members in the family Luteoviridae and its sequence can be more informative than other regions in the viral genome. Species specific RT-PCR assays targeting the same region were also developed for the detection of the six most widespread poleroviral species (Beet mild yellowing virus, Beet western yellows virus, Cucurbit aphid-borne virus, Carrot red leaf virus, Potato leafroll virus and Turnip yellows virus) in Greece and the collection of isolates.

A novel strategy for the determination of a rhabdovirus genome and its application to sequencing of Eggplant mottled dwarf virus.

A novel strategy employing the rhabdovirus untranslated conserved intergenic regions was developed and applied successfully for the determination of the complete nucleotide sequence of Eggplant mottled dwarf virus (EMDV). The EMDV genome contains seven open reading frames with the same organization as Potato yellow dwarf virus (PYDV), the type species of the genus Nucleorhabdovirus. These two species encode five core genes [nucleocapsid (N), phosphoprotein (P), matrix (M), glycoprotein (G), and the polymerase (L)] like other viruses of the genus and an additional one (X), located between N and P, giving rise to a protein with currently unknown function.

Generic detection and differentiation of tobamoviruses by a spot nested RT-PCR-RFLP using dI-containing primers along with homologous dG-containing primers.

A spot nested RT-PCR-RFLP method to detect and identify all members of the Tobamovirus genus is described. It involves a one-step RT-PCR, in which the combination of degenerate deoxyinosine (dI)-substituted primers amplified part of the polymerase region of tobamoviruses, followed by a nested PCR amplification that increased specificity and sensitivity of detection. Virus species differentiation was achieved by subsequent restriction enzyme analysis.