Molecular characterization of Hellenic variants of Apple scar skin viroid and Pear blister canker viroid in pome fruit trees

Apple scar skin viroid (ASSVd) and Pear blister canker viroid (PBCVd) are members of the genus Apscaviroid (family Pospiviroidae). In order to study the nucleotide sequence and secondary structure of Hellenic variants of these viroids, a large number of collected samples were initially screened by imprint hybridization; then ASSVd and PBCVd positive samples were assayed for the viroids by RT-PCR. Total RNA extracts were reverse-transcribed and amplified by polymerase chain reaction using two different specific primer pairs for each viroid. Purified RT-PCR products were directly sequenced or cloned into the pGEM-T and pCR® II vectors and then sequenced. Fourteen Hellenic full length ASSVd variants from 3 apple, 3 wild apple (Malus sylvestris), 1 wild pear (Pyrus amygdaliformis) and 3 pear trees are 330-335 nucleotides long. They differ from the reference sequences of ASSVd (ASSCS and Y00435) at 15-29 and 3-36 sites, respectively. Fifteen nucleotide changes (differences from ASSCS) are common among all Hellenic variants. Hellenic ASSVd variants share high identity (97-100%) with ASSVd isolates from Asian apples. Three Hellenic variants, deriving from different hosts and areas, are identical with each other (wild apple and apple from Pella [Macedonia] and pear from Achaia [Peloponnesus]) and with another group of 3 apple variants from China (Liaoning, AM1 and B-9). Sixteen full length Hellenic PBCVd variants from 12 trees (4 apples, 1 wild apple, 5 pears, 1 wild pear and 1 quince) are 314-316 nucleotides long. There are 6-50 nucleotide changes among all Hellenic variants and the prototype PBCVd isolate (NC001830). Twenty-two (22) changes are identical among the majority of the Hellenic variants, regardless of origin, and 28-35 changes occur in PBCVd sequences obtained from apple and wild apple samples. In addition, 2 Hellenic PBCVd variants are 97-98% homologous to some Australian and European (Bosnian) PBCVd pear isolates, whereas the remaining 14 share 86-94% identity with Australian PBCVd isolates from pear, quince and Japanese pear (Pyrus pyrifolia). This is the first detailed molecular study of ASSVd and PBCVd in Hellenic cultivated and wild pome fruit trees.Keywords: ASSVd, PBCVd, pome fruit, molecular characterizatio

Pospiviroidae viroids in naturally infected stone and pome fruits in Greece

Viroid research on pome and stone fruit trees in Greece is important, as it seems that such viroids are widespread in the country and may cause serious diseases. Our research dealt with three Pospiviroidae species infecting pome and stone fruit trees, namely Apple scar skin viroid (ASSVd), Pear blister canker viroid (PBCVd) and Hop stunt viroid (HSVd). Tissue-print hybridization, reverse transcription-polymerase chain reaction (RT-PCR), cloning and sequencing techniques were successfully used for the detection and identification of these viroids in a large number of pome and stone fruit tree samples from various areas of Greece (Peloponnesus, Macedonia, Thessaly, Attica and Crete). The 58 complete viroid sequences obtained (30 ASSVd, 16 PBCVd and 12 HSVd) were submitted to the GenBank. Our results showed the presence of ASSVd in apple, pear, wild apple (Malus sylvestris), wild pear (Pyrus amygdaliformis) and sweet cherry; HSVd in apricot, peach, plum, sweet cherry, bullace plum (Prunus insititia), apple and wild apple; and PBCVd in pear, wild pear, quince, apple and wild apple. This research confirmed previous findings of infection of Hellenic apple, pear and wild pear with ASSVd, pear, wild pear and quince with PBCVd and apricot with HSVd. Our findings also revealed for the first time the natural mixed infection of apple and wild apple with (ASSVd+PBCVd+HSVd), of apple and pear with (ASSVd+PBCVd), and of wild apple with (ASSVd+HSVd), as well as the natural infection of Hellenic sweet cherry, peach, bullace plum and plum with HSVd. To our knowledge, this is the first published report of detecting HSVd and PBCVd in infected apple and wild apple, and ASSVd in sweet cherry. Keywords: ASSVd, PBCVd, HSVd, stone fruit, pome fruit, Greec

First report and molecular analysis of Apple scar skin viroid in sweet cherry

Apple scar skin viroid (ASSVd) is a serious pathogen of pome fruits. Recently, it has been reported in Chinese apricot and Chinese peach. In the context of our research on fruit tree viroids in Greece, ASSVd was initially detected in a sweet cherry tree cv ‘Tragana Edessis’ from Florina (Macedonia) by RT-PCR and this finding was confirmed by direct sequencing. This tree is located at the edge of a newly established apple orchard, along with other sweet cherry and wild cherry (Prunus avium) trees. In order to verify this interesting finding, we examined for ASSVd four sweet cherry trees, two wild cherry trees and their neighboring apple trees in the same orchard.The examination was done by imprint hybridization using an ASSVd-specific DIG-labelled probe at stringent hybridization conditions and by RT-PCR using two different ASSVd specific primer pairs. We obtained ASSVdpositive results for all 6 cherry trees. No ASSVd was detected in the apple trees of the orchard. Purified ASSVdpositive RT-PCR products from the cherries were directly sequenced or cloned into the pGEM-T vector and then sequenced. ASSVd sequences were obtained from 5 trees. These sequences are 327-340 nucleotides long and share 96-99% identity with ASSVd isolates from Asian (Indian) apples. These results are similar to our data for other ASSVd variants from cultivated and wild pome fruit trees in Greece. The cherry ASSVd variants differ from the ASSCS prototype isolate of ASSVd at 18-29 sites. There are 15 nucleotide changes (differences from ASSCS) common to all Hellenic ASSVd variants, including cherry and pome fruit tree variants. There are no cherry-specific nucleotide changes in the ASSVd sequences obtained. To our knowledge, this is the first published report of natural infection of cherry by ASSVd. Keywords: ASSVd, cherry, molecular analysis, Hellenic sequence

Improvement of the reverse transcription loop mediated isothermal amplification (RTLAMP) method for the detection of Peach latent mosaic viroid (PLMVd)

Peach latent mosaic viroid (PLMVd) is the most known peach viroid. Among the diagnostic techniques used for its detection, the most recent described being the reverse transcription loop-mediated isothermal amplification method (RT-LAMP). Several modifications were done on the basic protocol proposed by Boubourakas et al. (2009), additional experiments were preformed in order to further evaluate the method. Namely, the reaction time was further reduced and traces of leaf tissue, taken by a sterile toothpick, instead of tRNA were used as the intial material. Moreover, the AMV reverse transcriptase proved to be more effective than Thermoscript, while restriction enzyme analysis was performed on the RT-LAMP products in order to confirm that products had the respective sequences of the selected target. Finally, the extremely high efficiency and sensitivity of RT-LAMP proved to be sufficient for the detection of PLMVd in hosts other than peach. Keywords: PLMVd, RT-LAMP, peach, reverse transcriptase

A non-mechanically transmissible isometric virus associated with asteroid mosaic of the grapevine

Research Not

Molecular detection of Grapevine fleck virus-like viruses

Molecular reagents have been developed for virus-specific and simultaneous (virus-non-specific) detection of Grapevine fleck virus (GFkV) and allied viruses, ie. Grapevine asteroid mosaic-associated virus (GAMaV) and Grapevine red globe virus (GRGV). Degenerate primers designed on nucleotide sequences of the RNA-dependent RNA polymerase (RD) and methyltransferase (MTR) domains of the GFkV genome, were able to give amplification products of the expected size from total nucleic acid extracts of:vines infected with GFkV, GAMaV, and GRGV;a Californian grapevine accession infected by a marafi-like virus;Greek grapevine accessions infected by an unidentified agent that induced symptoms reminiscent of those elicited by GAMaV in Vitis rupestris.Degenerate primers designed on the nucleotide sequence of the helicase (HEL) domain of the GFLV genome recognized all the above viruses except for GAMaV and the unidentified Greek viral agent. RD primer set worked well also with crude grapevine cortical scrapings, thus constituting a useful universal reagent for the non-specific molecular identification of GFkV-like viruses in Vitis . The marafi-like virus from California was amplified by all sets of primers, but was recognized only by the GRGV-specific probe, suggesting that it is a likely isolate of GRGV: Likewise, the unidentified virus from Greek vines shared sequence homology with GFkV and allied viruses (GAMaV and GRGV) but exhibited differences relevant enough that call for further investigations to establish its taxonomic position. While GRGV was identified, though with a very low incidence, in some 11 southern Italian grapevine cultivars, no evidence was obtained for infection by GAMaV in any of 50 cultivars analyzed.

Distribution Of Peach Latent Mosaic Viroid In Commercial Orchards Of Peach In The North Of Tunisia

peer reviewe

Molecular features of new Peach Latent Mosaic Viroid variants suggest that recombination may have contributed to the evolution of this infectious RNA.

peer reviewe

Aberrant Herpesvirus-Induced Polyadenylation Correlates With Cellular Messenger RNA Destruction

Inhibition of host cell gene expression by the human herpesvirus KSHV occurs via a novel mechanism involving polyadenylation-linked RNA turnover