Evaluation of the RNA Silencing Suppression Activity of Three Cherry Virus F-Encoded Proteins.

Cherry virus F (CVF) is a newly emerged sweet cherry virus. CVF has been identified in a small number of countries and it has not been associated with discrete symptomatology. RNA silencing is a natural defense mechanism of plants against invaders that degrades viral RNA in a sequence-specific manner. As a counter-defense, plant viruses encode one or more RNA silencing suppressors (RSSs) interfering with the silencing pathway via several mechanisms. To identify putative RSSs, the three proteins (MP, CPL, CPS) encoded by the RNA2 of CVF were selected and separately cloned into the binary vector pART27. The clones were used for transient expression experiments in Nicotiana benthamiana leaves, using co-agroinfiltration with a GFP-expressing vector. In both CPL and CPS, a rapid decrease in fluorescence was recorded, comparable to the negative control, whereas the MP of CVF retained the GFP's fluorescence for a few days longer even though this was observed in a small number of infiltrated leaves. Further experiments have shown that the protein was not able to inhibit the cell-to-cell spread of the silencing signal; however, a putative interference with systemic silencing was recorded especially when the induction was carried out with double-stranded GFP RNA. Overall, our results indicate that the MP of CVF is putatively implicated in the suppression of RNA silencing, though further experimentation is needed to unveil the exact mode of action.

Identification of divergent isolates of cherry latent virus 1 in Greek sweet cherry orchards.

In this study, samples collected from eight sweet cherry trees in northern Greece were analyzed by high-throughput sequencing for the presence of viruses. Bioinformatic analysis revealed the presence of divergent isolates of cherry latent virus 1 (CLV-1), a recently identified trichovirus in a sweet cherry accession imported into the USA from the Republic of Georgia. The complete genome sequences of seven CLV-1 isolates were determined, and phylogenetic analysis indicated that they belonged to a separate clade from the previously characterized Georgian isolate. A small-scale survey confirmed the presence of CLV-1 in 47 out of 151 sweet cherry samples tested, and partial sequencing of 15 isolates showed a high degree of nucleotide sequence similarity among them.

Molecular Characterization of the Coat Protein Gene of Greek Apple Stem Pitting Virus Isolates: Evolution through Deletions, Insertions, and Recombination Events.

A RT-PCR assay developed to amplify the full coat protein (CP) gene of apple stem pitting virus (ASPV) was evaluated using 180 Greek apple and pear samples and showed a broad detection range. This method was used to investigate the presence of ASPV in quince in Greece and showed a high incidence of 52%. The sequences of 14 isolates from various hosts with a distinct RFLP profile were determined. ASPV population genetics and the factors driving ASPV evolution were analyzed using the Greek ASPV sequences, novel sequences from Brazilian apple trees and Chinese botanical Pyrus species, and homologous sequences retrieved from GenBank. Fourteen variant types of Greek, Brazilian and botanical isolates, which differ in CP gene length and presence of indels, were identified. In addition, these analyses showed high intra- and inter-group variation among isolates from different countries and hosts, indicating the significant variability present in ASPV. Recombination events were detected in four isolates originating from Greek pear and quince and two from Brazilian apples. In a phylogenetic analysis, there was a tendency for isolates to cluster together based on CP gene length, the isolation host, and the detection method applied. Although there was no strict clustering based on geographical origin, most isolates from a given country tended to regroup in specific clusters. Interestingly, it was found that the phylogeny was correlated to the type, position, and pattern of indels, which represent hallmarks of specific lineages and indicate their possible role in virus diversification, rather than the CP size itself. Evidence of recombination between isolates from botanical and cultivated species and the clustering of isolates from botanical species and isolates from cultivated species suggest the existence of a possible undetermined transmission mechanism allowing the exchange of ASPV isolates between the cultivated and wild/ornamental hosts.

Identification and Sequence Analysis of a Novel Ilarvirus Infecting Sweet Cherry.

In the present study, we utilized high throughput and Sanger sequencing to determine the complete nucleotide sequence of a putative new ilarvirus species infecting sweet cherry, tentatively named prunus virus I (PrVI). The genome of PrVI is comprised of three RNA segments of 3474 nt (RNA1), 2911 nt (RNA2), and 2231 nt (RNA3) and features conserved motifs representative of the genus Ilarvirus. BlastN analysis revealed 68.1-71.9% nt identity of PrVI with strawberry necrotic shock virus (SNSV). In subsequent phylogenetic analysis, PrVI was grouped together with SNSV and blackberry chlorotic ringspot virus (BCRV), both members of subgroup 1 of ilarviruses. In addition, mini-scale surveys in stone fruit orchards revealed the presence of PrVI in a limited number of sweet cherries and in one peach tree. Overall, our data suggest that PrVI is a novel species of the genus Ilarvirus and it consists the fifth member of the genus that is currently known to infect Prunus spp.

Interplay of Cucurbit Yellow Stunting Disorder Virus With Cucurbit Chlorotic Yellows Virus and Transmission Dynamics by Bemisia tabaci MED.

Cucurbit chlorotic yellows virus (CCYV) and cucurbit yellow stunting disorder virus (CYSDV) are two closely related criniviruses that often coinfect cucurbits and are associated with cucurbit yellows disease. Both viruses are distributed worldwide and are transmitted in a semipersistent manner by the whitefly vectors Bemisia tabaci MED or MEAM1. The major goal of this study was to provide insight into the interaction of CCYV and CYSDV in cucumber and to study the effect on transmission by B. tabaci MED. The titers of both viruses were estimated in single- and dually infected cucumber plants via reverse transcription PCR assays. In mixed infections, the accumulation of both viruses was significantly decreased. When B. tabaci MED adults were placed on cucumber infected with both viruses, their simultaneous transmission efficiency was significantly higher, whereas transmission efficiency of each individual virus was low. Moreover, nonviruliferous whiteflies preferentially settled on crinivirus-infected cucumber plants, whereas viruliferous whiteflies were attracted by healthy cucumber plants. Finally, the titer of both viruses was calculated in five commercial cucumber hybrids, followed by subsequent transmission experiments. Our results show that although the titers of CYSDV and CCYV were significantly lower in mixed infections in cucumbers, their simultaneous transmission increased.

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. The amplification efficiency of the duplex assays reached 91.1-95.8%, while the linear range of quantification was from 20 to 2 × 107 RNA/linearized plasmid transcripts for PLMVd and ESFY phytoplasma, 40 to 4 × 107 RNA transcripts for ACLSV, PPV and PDV, and 102 to 108 RNA transcripts for PNRSV, respectively. The duplex RT-qPCR assays, which were validated using both characterized isolates from all pathogens and field samples from Prunus species in Northern Greece, exhibited a broad detection range. Overall, the developed methods comprise useful tools that could be applied for the simultaneous and reliable detection of graft-transmissible pathogens in certification programs of Prunus spp.

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. Ten-fold serial dilutions of in vitro synthesized RNA transcripts were applied for the construction of standard curve. The amplification efficiency of the assay was 93.8% and the linear range of quantification was from 40 up to 4 × 108 RNA copies. The real time RT-PCR was successfully tested with a collection of genetically diverse isolates with different geographical origin belonging to seven PPV strains. The present method is proposed as a useful tool for various basic or applied research studies of PPV as well as for routine testing of plant material during phytosanitary control or in certification schemes of Prunus species.

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. Primers and a TaqMan probe were designed, using conserved regions of the capsid protein gene. Detection range was evaluated using several divergent viral isolates. The amplification efficiency of the method was estimated at 96.7%, whereas the detection limit was about 100 RNA copies. The protocol was applied in the study of virus fluctuation within leaves and phloem tissue throughout the year and the best periods to test and plant tissues to sample were determined. Comparative analysis of this method with a previously published nested RT-PCR revealed the higher analytical and diagnostic sensitivity of the new test, making it a reliable tool that can be used in routine testing and certification programs.

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. Sequence similarity and phylogenetic analysis highlighted the common ancestry of the three poleroviruses (PeVYV, PeYV, and Pepper yellow leaf curl virus [PYLCV]) currently reported to be associated with PYD, even though significant genetic differences were identified among them, especially in the C-terminal region of P5 and the 3' noncoding region. Most of the differences observed can be attributed to a modular type of evolution, which produces mosaic-like variants giving rise to these different poleroviruses Overall, similar to other polerovirus-related diseases, PYD is caused by at least three species (PeVYV, PeYV, and PYLCV) belonging to this group of closely related pepper-infecting viruses.

Genetic variation of eggplant mottled dwarf virus from annual and perennial plant hosts.

The genetic diversity of eggplant mottled dwarf virus (EMDV), a member of the family Rhabdoviridae, was studied using isolates collected from different herbaceous and woody plant species and remote geographic areas. Sequences corresponding to the N, X, P, Y, M and G ORFs as well as the untranslated regions (UTRs) between ORFs were determined from all isolates. Low genetic diversity was found in almost all genomic regions studied except for the X ORF and the UTRs, which were more variable, while interestingly, an EMDV isolate from caper possessed a truncated G gene sequence. Furthermore, low d N /d S ratios, indicative of purifying selection, were calculated for all genes. Phylogenetic analysis showed that the EMDV isolates clustered in three distinct subgroups based on their geographical origin, with the exception of one subgroup that consisted of isolates from northern Greece and Cyprus. Overall, the level of genetic diversity of EMDV differed between seed- and asexually propagated plants in our collection, and this could be related to the mode of transmission.

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. Analysis of the NGS data revealed the presence of various known grapevine viruses and viroids as well as a hitherto uncharacterized DNA virus. The circular genome of the new virus was fully reassembled. It is 6988 nts long and includes 4 open reading frames (ORFs). ORF1, ORF2 and ORF4 code for proteins with unknown functions while ORF3 encodes a polyprotein with motifs related to the replication, encapsidation and movement of the virus. Phylogenetic analysis classified the novel virus within the genus Badnavirus, with closest relationship to Fig badnavirus 1. Further studies showed that the new badnavirus is closely related with the RLD disease and the provisional name grapevine Roditis leaf discoloration-associated virus (GRLDaV) is proposed. Our findings extend the number of DNA viruses identified in grapevine, further drawing attention to the potential importance of this virus group on grapevine pathology.

Control of viruses infecting grapevine.

Grapevine is a high value vegetatively propagated fruit crop that suffers from numerous viruses, including some that seriously affect the profitability of vineyards. Nowadays, 64 viruses belonging to different genera and families have been reported in grapevines and new virus species will likely be described in the future. Three viral diseases namely leafroll, rugose wood, and infectious degeneration are of major economic importance worldwide. The viruses associated with these diseases are transmitted by mealybugs, scale and soft scale insects, or dagger nematodes. Here, we review control measures of the major grapevine viral diseases. More specifically, emphasis is laid on (i) approaches for the production of clean stocks and propagative material through effective sanitation, robust diagnosis, as well as local and regional certification efforts, (ii) the management of vectors of viruses using cultural, biological, and chemical methods, and (iii) the production of resistant grapevines mainly through the application of genetic engineering. The benefits and limitations of the different control measures are discussed with regard to accomplishments and future research directions.

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. 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.

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. 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.

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. 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.

Viruses of the genus Allium in the Mediterranean region.

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.

Cydonia japonica, Pyrus calleryana and P. amygdaliformis: three new ornamental or wild hosts of Apple stem pitting virus.

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.

Rapid discrimination of Tomato chlorosis virus, Tomato infectious chlorosis virus and co-amplification of plant internal control using real-time RT-PCR.

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.

Generic and species-specific detection of viruses belonging to an evolutionary distinct lineage within the Ampelovirus genus.

A nested RT-PCR was developed, that allows the generic detection of a subgroup of genetically related viruses with a distinct evolutionary history within the genus Ampelovirus. Members of this lineage are Grapevine leafroll associated virus-4, -5, -6, -9 and two isolates (GLRaV-De and GLRaV-Pr) that have been recently characterized and represent new species. The method involves a one step RT-PCR for the generic detection of Closteroviridae species using degenerate primers that target the HSP70h gene followed by a nested PCR, which detects all virus-members of the lineage and differentiates them from the other grapevine closteroviruses. The 490 bp nested PCR amplicons, corresponding to a phylogenetically informative region, can be sequenced directly to obtain initial genetic information for their partial characterization and rapid classification. Additional primers were designed and successfully used for the specific detection of GLRaV-4, -5, -6, -Pr and -De on respective single or multiplex nested PCR assays. The application of a ramped annealing thermal profile in the nested PCR allowed all amplifications to run in parallel. The developed detection scheme is proposed as a tool that can be used for the enrichment of sequence information of known and uncharacterized ampeloviruses, classified within this lineage, enabling their selective amplification in mixed Closteroviridae virus infections.