The Elimination of Viroids through In Vitro Thermotherapy and a Meristem Tip Culture from a New Limonime Hybrid (Citrus x limon var. limon (L.) Burm. f. x Citrus latifolia var. latifolia).

Viruses and viroids pose a significant challenge in citriculture, and their control is crucial for plant health. This study evaluated the effectiveness of in vitro thermotherapy combined with a meristem tip culture for eliminating citrus exocortis viroid (CEVd) and hop stunt viroid (HSVd) from a new limonime hybrid (Citrus x limon var. limon x Citrus latifolia var. latifolia). The elimination success was confirmed by RT-PCR assays. The in vitro elimination rate for CEVd during the shoot proliferation stage (43%) was higher than for HSVd (21%). Accordingly, in the subsequent rooting stage, the in vitro elimination rate for CEVd (50%) was higher than for HSVd (33%). Successful CEVd and HSVd eradication at a 100% rate was confirmed in the ex vitro acclimatized plants in the greenhouse. The study also established an efficient micropropagation protocol. The optimal treatment for in vitro shoot induction was 0.5-2 mg L-1 benzyladenine (BA) + 0.5 mg L-1 gibberellic acid (GA3) + 0.25 mg L-1 naphthalene acetic acid (NAA), while for shoot elongation, it was 0.5 mg L-1 BA + 0.5 mg L-1 kinetin (KIN) + 0.5 mg L-1 GA3 + 0.25 mg L-1 NAA. Rooting was best promoted by 1 mg L-1 NAA. This study provides valuable insights for the mass production of viroid-free propagation material in this new lemon x lime hybrid, contributing to the conservation of genetic resources in citrus breeding programs through the combined application of in vitro thermotherapy and an in vitro meristem tip culture, a novel and highlighted achievement reported for the first time in this study.

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.

Identification and complete genome sequencing of a divergent olive virus T isolate and an olive leaf yellowing-associated virus isolate naturally infecting olive trees in Greece.

Several new full genome sequences of olive viruses came to light recently via high-throughput sequencing (HTS) analysis. In this study, total RNA HTS analysis of two Greek olive trees revealed the presence of an olive virus T (OlVT) isolate and an olive leaf yellowing-associated virus (OLYaV) isolate. The full viral genome of OlVT isolate (50Ch) is composed of 6862 nucleotides encoding for three proteins (replicase, movement protein, and capsid protein) with typical betaflexiviruses' genomic features. However, both sequence and phylogenetic data analysis exhibited high levels of variability between 50Ch and the previously characterized OlVT isolates. In addition, the almost full genome of the Greek OLYaV isolate (OL2) was obtained, which is composed of 16,693 nucleotides encoding for 11 open reading frames (ORFs) and shares common genomic features with the recently characterized OLYaV isolates from Spain and Brazil. Sequence and phylogenetic analysis revealed high similarity between these three isolates. Due to problems encountered with the detection of both viruses, new nested RT-PCR assays were developed and applied. In addition, recombination events were observed in OlVT isolates (50Ch GR-168), thus highlighting the potential role of this mechanism in the evolution of the virus. This study is adding further knowledge to the limited information available about these recently characterized olive infecting viral pathogens and highlights their widespread distribution in Greece, one of the most important olive producing countries of the world.

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.

Complete genome sequence of a grapevine Roditis leaf discoloration-associated virus (GRLDaV) variant from South Africa.

High-throughput sequencing (HTS) was used to construct the virome profile of an old grapevine-leafroll-diseased grapevine (Vitis vinifera). De novo assembly of HTS data showed a complex infection, including a virus sequence with similarity to viruses of the genus Badnavirus, family Caulimoviridae. The complete genome sequence of this virus consists of 7090 nucleotides and has four open reading frames (ORFs). Genome organisation and phylogenetic analysis identify this virus as a divergent variant of grapevine Roditis leaf discoloration-associated virus (GRLDaV) with 90% nucleotide sequence identity to isolate w4 (NC_027131). This is the first genome sequence of a South African variant of GRLDaV.

First report of grapevine virus H (GVH) in grapevine in Greece.

Grapevine virus H (GVH) is a member of the genus Vitivirus in the family Betaflexiviridae (subfamily Trivirinae, order Tymovirales) that infects grapevine (Candresse et al., 2018). GVH was first identified in a symptomless grapevine of an unknown cultivar from Portugal in 2018 (Candresse et al. 2018), and since then the virus has been reported only from California (Diaz­Lara et al. 2019). Several vitiviruses have been detected in Greek vineyards (Avgelis and Roubos 2000; Dovas and Katis 2003a; 2003b; Panailidou et al. 2019; Lotos et al. 2020), but no information was available on the presence of GVH. In the fall of 2020, in order to investigate the virome of a commercial vineyard of the cultivar Assyrtiko in northern Greece, a composite sample was made of leaves and petioles from nine vines exhibiting leafroll disease symptoms. Total RNA was extracted from the composite sample according to the protocol of White et al. (2008) and subjected to rRNA depletion, library construction (TruSeq Stranded Total RNA kit), and high-throughput sequencing (HTS) in a NovaSeq6000 platform (Illumina Inc.) at Macrogen (Korea). The resulting ~42 million 101-nt paired-end reads were analyzed in Geneious Prime 2020, and the assembled de novo contigs were subjected to a local BLASTn search, which revealed the presence of 18 grapevine infecting viruses and viroids, among which also a GVH-like contig (GeA-9). GeA-9 was 7,404 nucleotides (nt) long, covering 99.4% of the full virus genome and shared 98.2 % nt identity with a GVH isolate from the USA (MN716768). To confirm the presence of GVH, the nine samples of the cultivar Assyrtiko, used initially to produce the composite sample for HTS analysis, were tested individually by RT-PCR, using the primers GVH_F_2504 (5'-CTGCTTCGCTGAACATATGC-3') and GVH_R_2835 (5'-ATCATTRTGATCGAGAGAGTAGTG-3') that amplify a 331-nt segment of ORF1. GVH was detected in five out of the nine tested samples and one of these was reamplified and subjected to Sanger sequencing. The fragment of ORF1 obtained by Sanger sequencing (MW460005) was 97.5% identical to the nucleotide sequence of the corresponding GVH-like de novo contig (GeA-9) from HTS analysis and it shared 97.2% nt identity with GVH sequences reported from Portugal and USA, respectively (NC_040545 and MN716768), confirming the presence of GVH in the tested samples. This is the first report of GVH in grapevine in Greece, thus further increasing the number of vitiviruses known to infect Greek vineyards and also expanding the number of geographic locations in which GVH is recorded so far.

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.

Specific Real-Time PCR for the Detection and Absolute Quantitation of Grapevine Roditis Leaf Discoloration-Associated Virus, an EPPO Alert Pathogen.

Grapevine Roditis leaf discoloration-associated virus (GRLDaV) is an emerging grapevine pathogen included in the European and Mediterranean Plant Protection Organization (EPPO) alert list due to its ability to damage grapevine crops and cause production losses. This work aimed to develop a specific and reliable diagnostic tool that would contribute to preventing the spread of this pathogen. Therefore, a TaqMan real-time quantitative PCR was developed. The method was validated according to EPPO guidelines showing a high degree of analytical sensitivity, analytical specificity, selectivity, and repeatability and reproducibility. The sensitivity of this method is much higher than the sensitivity reached by previously reported methods even when tested in crude extracts, which could allow rapid testing by avoiding nucleic acid extraction steps. The method was also able to detect GRLDaV isolates from all the geographic origins reported so far, despite their high degree of genetic diversity. In addition, this new technique has been successfully applied for the quantitative detection of GRLDaV in plant material and two mealybug species, Planococcus citri and Pseudococcus viburni. In conclusion, the methodology developed herein represents a significant contribution to the diagnosis and control of this emerging pathogen in grapevine.

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.

Cucurbit chlorotic yellows virus p22 is a suppressor of local RNA silencing.

RNA silencing is a major antiviral mechanism in plants, which is counteracted by virus-encoded proteins with silencing suppression activity. ORFs encoding putative silencing suppressor proteins that share no structural or sequence homology have been identified in the genomes of four criniviruses. In this study, we investigated the RNA silencing suppression activity of several proteins encoded by the RNA1 (RdRp, p22) and RNA2 (CP, CPm and p26) of cucurbit chlorotic yellows virus (CCYV) using co-agroinfiltration assays on Nicotiana benthamiana plants. Our results indicate that p22 is a suppressor of local RNA silencing that does not interfere with cell-to-cell movement of the RNA silencing signal or with systemic silencing. Furthermore, comparisons of the suppression activity of CCYV p22 with that of two other well-known crinivirus suppressors (CYSDV p25 and ToCV p22) revealed that CCYV p22 is a weaker suppressor of local RNA silencing than the other two proteins. Finally, a comparative sequence analysis of the p22 genes of seven Greek CCYV isolates was performed, revealing a high level of conservation. Taken together, our research advances our knowledge about plant-virus interactions of criniviruses, an emergent group of pathogens that threatens global agriculture.

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.

Recent Advances on Detection and Characterization of Fruit Tree Viruses Using High-Throughput Sequencing Technologies.

Perennial crops, such as fruit trees, are infected by many viruses, which are transmitted through vegetative propagation and grafting of infected plant material. Some of these pathogens cause severe crop losses and often reduce the productive life of the orchards. Detection and characterization of these agents in fruit trees is challenging, however, during the last years, the wide application of high-throughput sequencing (HTS) technologies has significantly facilitated this task. In this review, we present recent advances in the discovery, detection, and characterization of fruit tree viruses and virus-like agents accomplished by HTS approaches. A high number of new viruses have been described in the last 5 years, some of them exhibiting novel genomic features that have led to the proposal of the creation of new genera, and the revision of the current virus taxonomy status. Interestingly, several of the newly identified viruses belong to virus genera previously unknown to infect fruit tree species (e.g., Fabavirus, Luteovirus) a fact that challenges our perspective of plant viruses in general. Finally, applied methodologies, including the use of different molecules as templates, as well as advantages and disadvantages and future directions of HTS in fruit tree virology are discussed.

High-Throughput Sequencing Reveals Further Diversity of Little Cherry Virus 1 with Implications for Diagnostics.

Little cherry virus 1 (LChV1, Velarivirus, Closteroviridae) is a widespread pathogen of sweet or sour cherry and other Prunus species, which exhibits high genetic diversity and lacks a putative efficient transmission vector. Thus far, four distinct phylogenetic clusters of LChV1 have been described, including isolates from different Prunus species. The recent application of high throughput sequencing (HTS) technologies in fruit tree virology has facilitated the acquisition of new viral genomes and the study of virus diversity. In the present work, several new LChV1 isolates from different countries were fully sequenced using different HTS approaches. Our results reveal the presence of further genetic diversity within the LChV1 species. Interestingly, mixed infections of the same sweet cherry tree with different LChV1 variants were identified for the first time. Taken together, the high intra-host and intra-species diversities of LChV1 might affect its pathogenicity and have clear implications for its accurate diagnostics.

Variability Studies of Two Prunus-Infecting Fabaviruses with the Aid of High-Throughput Sequencing.

During their lifetime, perennial woody plants are expected to face multiple infection events. Furthermore, multiple genotypes of individual virus species may co-infect the same host. This may eventually lead to a situation where plants harbor complex communities of viral species/strains. Using high-throughput sequencing, we describe co-infection of sweet and sour cherry trees with diverse genomic variants of two closely related viruses, namely prunus virus F (PrVF) and cherry virus F (CVF). Both viruses are most homologous to members of the Fabavirus genus (Secoviridae family). The comparison of CVF and PrVF RNA2 genomic sequences suggests that the two viruses may significantly differ in their expression strategy. Indeed, similar to comoviruses, the smaller genomic segment of PrVF, RNA2, may be translated in two collinear proteins while CVF likely expresses only the shorter of these two proteins. Linked with the observation that identity levels between the coat proteins of these two viruses are significantly below the family species demarcation cut-off, these findings support the idea that CVF and PrVF represent two separate Fabavirus 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.