[Show abstract][Hide abstract] ABSTRACT: 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 × 108 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. The recovery rates of RNA and the effect of co-extracted inhibitors revealed that methods involving PVPP and phenol–chloroform extraction were the most efficient. These modifications were necessary for processing samples containing high phenolic and polysaccharide compounds such as woody plants. The developed EMDV detection protocol was successfully applied in forty naturally infected woody and herbaceous plants belonging to six different species. The protocol comprises a useful method for low-cost detection of ssRNA viruses in diverse plant tissues.
[Show abstract][Hide abstract] ABSTRACT: Little cherry virus 1 (LChV-1), a member of the recently proposed genus Velarivirus is a sweet cherry pathogen which has been lately reported to infect also other Prunus species and is presumably associated with various plant disorders. In this work we studied the incidence of the virus on its putative hosts and tried to understand the mechanisms driving its evolution. Due to problems encountered with the LChV-1 detection, a new nested RT-PCR assay was developed and applied herein. The virus was found to be prevalent in cherry plantations in Greece and only occasionally it was detected in other Prunus species. Sequences corresponding to the partial RdRp, HSP70h and CP were determined from Greek LChV-1 isolates originating from different hosts and used along with already published homologous genomic regions from other isolates. Phylogenetic analysis of the three genes revealed the segregation of four evolutionary distinct groups showing no host or geography-based clustering. Mean genetic distances among the four groups were high with the CP region showing the highest divergence, though intra-group variability levels were low. Nevertheless, estimations of dN/dS for the partial RdRp, HSP70h and CP indicated that these genomic regions are under negative selection pressure. Interestingly, a recombination event was identified at the 3΄end of the RdRp on a Greek virus isolate thus highlighting the role of this mechanism in the evolutionary history of LChV-1.This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: N.Duran-Vila, J.Juarez, J.M.Arregui, M.I.Molins. 1987. Production of viroid-free grapevines by shoot-tip culture. IX Meeting of the International Council for the study of viruses and virus diseases of the grapevine. Israel, Septiembre 1987. Abstract P.18.
[Show abstract][Hide abstract] ABSTRACT: A large-scale survey was carried out to study the host range and genetic diversity of Apple chlorotic leaf spot virus (ACLSV) in various Rosaceae species, with a special emphasis on ornamentals and wild shrubs. Samples were tested by DAS-ELISA using two different antisera, and RT-PCR amplification of part of the CP gene. There was generally a poor correlation between the results obtained with the two sets of serological reagents and between serological and molecular detection assays. Using a nested RT-PCR assay developed here, ACLSV was found to be widespread among cultivated, ornamental and wild species of the Rosaceae. The virus was detected for the first time in plum, wild cherry, Crataegus monogyna, Prunus spinosa and Prunus cerasifera in Greece. Sequences of a part of the CP encoding gene and the 3′ untranslated region from ACLSV isolates originating from various wild species and ornamentals were compared to those of isolates from cultivated hosts, showing similar divergence levels. Further phylogenetic analysis using the sequenced region indicated that the isolates from wild or ornamental hosts were not more closely related to each other than to isolates from cultivated hosts. The possible role of different factors in the spread of ACLSV on cultivated, ornamental and wild species is discussed.
[Show abstract][Hide abstract] ABSTRACT: Cauliflower mosaic virus (CaMV) is a plant pararetrovirus with a double-stranded DNA genome. It is the type member of the genus Caulimovirus in the family Caulimoviridae. CaMV is transmitted by sap inoculation and in nature by aphids in a semi-persistent manner. To investigate the patterns and timescale of CaMV migration and evolution, we sequenced and analyzed the genomes of 67 isolates of CaMV collected mostly in Greece, Iran, Turkey, and Japan together with nine published sequences. We identified the open-reading frames (ORFs) in the genomes and inferred their phylogeny. After removing recombinant sequences, we estimated the substitution rates, divergence times, and phylogeographic patterns of the virus populations. We found that recombination has been a common feature of CaMV evolution, and that ORFs I-V have a different evolutionary history from ORF VI. The ORFs have evolved at rates between 1.71 and 5.81×10(-4) substitutions/site/year, similar to those of viruses with RNA or ssDNA genomes. We found four geographically confined lineages. CaMV probably spread from a single population to other parts of the world around 400-500 years ago, and is now widely distributed among Eurasian countries. Our results revealed evidence of frequent gene flow between populations in Turkey and those of its neighboring countries, with similar patterns observed for Japan and the USA. Our study represents the first report on the spatial and temporal spread of a plant pararetrovirus.
PLoS ONE 01/2014; 9(1):e85641. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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. These isolates along with other characterized ones were used for the evaluation of the generic PCR's detection range. The developed assay efficiently amplified a 593bp RdRp fragment from 46 isolates of 10 different Polerovirus species. Phylogenetic analysis using the generic PCR's amplicon sequence showed that although it cannot accurately infer evolutionary relationships within the genus it can differentiate poleroviruses at the species level. Overall, the described generic assay could be applied for the reliable detection of Polerovirus infections and, in combination with the specific PCRs, for the identification of new and uncharacterized species in the genus.
Journal of virological methods 12/2013; · 2.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tomato chlorosis virus (ToCV) and Tomato infectious chlorosis virus (TICV) are two whitefly transmitted viruses which are classified in the genus Crinivirus of the family Closteroviridae. Both induce similar yellowing symptoms in tomato and are responsible for severe economic losses. ToCV is transmitted by Bemisia tabaci Gennadious, Trialeurodes vaporariorum Westwood and Trialeurodes abutilonea Haldeman, whereas TICV is transmitted only by T. vaporariorum. An extensive study was conducted during 2009-2012 in order to identify the virus species involved in tomato yellowing disease in Greece. Samples from tomato, other crops and weeds belonging to 44 species from 26 families were collected and analyzed using molecular methods. In addition, adult whiteflies were collected and analyzed using morphological characters and DNA markers. Results showed that TICV prevailed in tomato crops (62.5%), while ToCV incidence was lower (20.5%) and confined in southern Greece. ToCV was also detected in lettuce plants showing mild yellowing symptoms for the first time in Greece. Approximately 13% of the tested weeds were found to be infected, with TICV being the predominant virus with an incidence of 10.8%, whereas ToCV was detected only in 2.2% of the analyzed samples. These results indicate that the host range of TICV and ToCV in Greece is far more extensive than previously believed. T. vaporariorum was the most widespread whitefly species in Greece (80%), followed by B. tabaci (biotypes B and Q) (20%). Sequence analysis of the CP and CPm genes from Greek tomato and weed isolates of ToCV and TICV showed that even though both viruses have very wide host ranges their populations show very low molecular divergence.
[Show abstract][Hide abstract] ABSTRACT: 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. Furthermore, both EMDV and PYDV contain a gene (Y), inserted between P and M, which probably encodes the virus movement protein, in concordance with the rest of the plant-infecting rhabdoviruses. Phylogenetic analysis of the polymerase gene confirmed the classification of EMDV within the genus Nucleorhabdovirus and showed a close evolutionary relationship to PYDV. The novel sequencing strategy developed is a useful tool for the genome determination of yet uncharacterized rhabdoviruses.
[Show abstract][Hide abstract] ABSTRACT: The Bursa and Sakarya provinces in the south eastern part of Marmara region in Turkey [(39°.34 1) -41°.10 1 N, 28°.05 1 -30°.54 1 E] are very important fruit-growing areas. Leaf samples from 12 symptomless pear trees and six quince trees showing typical symptoms of quince fruit de-formation disease (Nemeth, 1986), including leaf chlorosis and curling, mosaic, vein banding, and deformation, were collected in June 2009 and tested for the presence of Apple stem pitting virus (ASPV), genus Foveavirus, family Betaflexiviridae. RT-PCR for the generic detection of foveaviruses was used with degenerate primers that target a conserved region of the RNA-dependent RNA poly-merase gene, followed by a nested PCR that amplifies a 312 bp ASPV-specific product (Mathioudakis et al., 2009). The virus was present in all pear and quince samples tested (GenBank accession Nos FN432827 and FN432828, re-spectively). Direct sequencing of two RT-PCR amplicons, one from pear and one from quince, confirmed the identi-fication of ASPV. The pear isolate (ASPV-Pe) showed 83.0% nucleotide sequence identity with a pear isolate of ASPV (accession No. FN386784) whereas the quince iso-late (ASPV-Qui) showed 84.0% nucleotide sequence iden-tity with an apple isolate of ASPV (accession No. FN386781). Nucleotide sequence comparison among AS-PV-Pe and ASPV-Qui isolates revealed a 78.7% similarity. To our knowledge, these findings represent the first report of ASPV in pear and quince orchards in northern Turkey.-tis N.I., 2009. Reliable RT-PCR detection of Apple stem pit-ting virus in pome fruits and its association with quince fruit deformation disease. Plant
[Show abstract][Hide abstract] ABSTRACT: Japanese quince, ornamental and wild pear symptomless samples were infected with Apple stem pitting virus (ASPV). Identification of ASPV was achieved by different PCR assays that amplified either the RNA polymerase or coat protein gene regions. For further confirmation, 312 bp amplicons within the polymerase gene were sequenced and compared with previously published ASPV sequences and additional sequences of isolates from ancient Italian cultivars. Comparison of the partial sequences isolated from wild/ornamental hosts and from cultivated species revealed significant divergence levels. Among the wild/ornamental isolates, the PCT88 isolate from Pyrus calleryana was the most divergent, having an amino acid deletion and incorporating a unique stretch of amino acids not present in any other isolate. Further to this preliminary partial sequence data, statistical analysis demonstrated that the isolates from wild or ornamental hosts were not more closely related to each other than to isolates from cultivated hosts. These results represent the first report of natural ASPV infection in these novel ornamental and wild Rosaceae hosts.
[Show abstract][Hide abstract] ABSTRACT: Allium species are economically important crops in the Mediterranean basin. Viruses are among the most important pathogens affecting their yield and especially those belonging to the genera Potyvirus, Carlavirus, and Allexivirus. Members of the genus Potyvirus are usually the most abundant and cause most of the damage induced. Nevertheless, coinfections with different viruses are not scarce, especially in garlic, and can have synergistic effects that lead to even greater crop losses. Vegetative propagation of alliums and the transmission of most of their viruses by arthropod vectors have significantly contributed to their wide dissemination in the Mediterranean region and elsewhere in the world. Here, we review the general biological and molecular features, the epidemiology, incidence, and methods of diagnosis of the most widespread allium viruses in the basin. Control measures are proposed depending on the mode of propagation of the various alliums, the epidemiology of their viruses and the cultivation procedures adapted by the Mediterranean farmers. The importance of the production and use of virus-free propagative material in order to combat viral diseases of allium crops is especially highlighted. A final discussion focuses on the main shortages identified in the research area of allium viruses, and proposals are made for putative future developments.
Advances in Virus Research 01/2012; 84:163-208. · 2.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tomato chlorosis virus (ToCV) and Tomato infectious chlorosis virus (TICV) (genus: Crinivirus, family: Closteroviridae) are two emergent whitefly-transmitted viruses that have been associated with yellowing symptoms of tomato crops during the last two decades. A real-time, one-step reverse transcription (RT) TaqMan(®) polymerase chain reaction (PCR) assay was developed and optimized for the multiplex detection of TICV, ToCV and an internal control of mitochondrion cytochrome oxidase subunit I (mtCOXI) gene from plants. The plant mtCOXI assay can be used as an internal control in at least 77 plant species from 28 different families. The one-step RT TaqMan PCR assay successfully detected and discriminated the two virus species in infected tomato plants, other host plants and their whitefly vectors. In direct comparison, the assay was approximately 10,000-fold and 100-fold more sensitive than conventional one-step RT-PCR and two-step nested RT-PCR, respectively. The increased sensitivity allowed the use of alternative template preparation methods that do not require RNA purification. The assay can be performed either by the direct addition of crude plant extract into the real-time reaction mixture or alternatively, the sap extract can be blotted on a positively charged nylon membrane, eluted and added in the reaction mixture. The developed assay allows the simple, fast and cost-effective testing of a large number of samples and can be easily applied in surveys and certification schemes.
Journal of virological methods 06/2011; 176(1-2):53-9. · 2.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The aim of this survey was to identify viruses infecting spinach (Spinacia oleracea L.) in the most important spinach-producing areas in Greece. A total of 1074 spinach samples were collected from eleven districts belonging to the prefectures of Thessaloniki, Chalkidiki and Imathia in northern Greece, and Evia in central Greece. Samples were tested by ELISA, mechanical inoculation onto indicator plants and immunoelectron microscopy. Beet western yellows virus (BWYV), Cucumber mosaic virus (CMV) and Turnip mosaic virus (TuMV) were identified in 13.5%, 7% and 5.4% of samples, respectively, infected samples being detected in all regions examined. This is the first record of CMV and TuMV infecting spinach in Greece, and the first report of BWYV occurrence in any crop nationwide. Surveys were also conducted to assess the potential reservoir hosts of BWYV, CMV and TuMV in weeds collected from spinach fields. All three viruses were detected among 125 samples tested by ELISA. TuMV prevailed as it occurred in 14.4% of all weed samples.
JOURNAL OF PLANT PATHOLOGY 01/2011; 93(2):389-395. · 0.77 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: During the past four decades, Tomato yellow leaf curl disease has become one of the major constraints in tomato production worldwide. In the Mediterranean basin, several isolates from two major Begomovirus species are involved in outbreaks and persistent epidemics. A real-time TaqMan PCR assay was developed and evaluated for the rapid and multiplex detection and differentiation of two begomoviruses often found in mixed infections in the region, Tomato yellow leaf curl virus (TYLCV) and Tomato yellow leaf curl Sardinia virus (TYLCSV). This assay was 1000-fold more sensitive than conventional PCR assays described previously, allowing the use of simple template preparation methods and eliminating the need for total nucleic acid purification. The viral DNA template was obtained by spotting sap extract derived from TYLCV or TYLCSV infected tissues on a nylon membrane or by directly using crude plant extracts in the real-time reaction cocktail. Preliminary results showed that this method can successfully detect and discriminate virus species from infected tomato, bean, pepper and different weed species obtained from the Mediterranean basin, the USA and Japan, allowing the simple, fast and cost-effective testing of a large number of samples in certification schemes. The assay can also be used for the detection of these two begomovirus species in their whitefly vector biotypes of the Bemisia tabaci (Gennadius) species group.
Journal of virological methods 02/2010; 165(2):238-45. · 2.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Abstract The phylogenetic relationships among Potato virus Y (PVY) isolates from northern and southern Greece were investigated. A large part of coat protein gene of 49 tobacco isolates and three from pepper was examined. The analysis showed that all 52 isolates consisted of 34 distinct haplotypes, with only one haplotype found in both northern and southern regions. The southern population was more diverse than that from the north. The phylogenetic analyses of the Greek haplotypes alone or in combination with isolates from other countries using the maximum likelihood method classified unambiguously almost all the haplotypes examined. Nine tobacco haplotypes from the south were classified as C-like (particularly C1), whereas 22 haplotypes from tobacco and two from pepper from both north and south were classified as N-like. One tobacco haplotype from the south was found recombinant between N-like and C1 lineages. The pattern of molecular evolution was examined using the fixed-effects likelihood and the single-likelihood ancestor counting methods. The analysis indicated that the evolution of PVY isolates appeared to be conservative (purifying selection and neutral evolution). These findings are discussed in relation to the introduction of PVY in the tobacco crop in Greece and the between region dispersal. A scenario of multiple introductions of PVY isolates in north and south Greece from different genetic pools and low or nil between region spread of the virus isolates was proposed.
Journal of Phytopathology 01/2010; 158:73-80. · 1.00 Impact Factor