A subset of highly responsive transcription factors upon tomato infection by pepino mosaic virus.

Plants have evolved well-tuned surveillance systems, including complex defence mechanisms, to constrain pathogens. TFs are master regulators of host molecular responses against plant pathogens. While PepMV constitutes a major threat to the global tomato production, there is still a lack of information on the key TFs that regulate host responses to this virus. A combinatorial research approach was applied relying on tomato transcriptome analysis, RT-qPCR validation, phylogenetic classification, comparative analysis of structural features, cis-regulatory element mining and in silico co-expression analysis to identify a set of 11 highly responsive TFs involved in the regulation of host responses to PepMV. An endemic PepMV isolate, generating typical mosaic symptoms, modified expression of ca. 3.3% of tomato genes, resulting in 1,120 DEGs. Functional classification of 502 upregulated DEGs revealed that photosynthesis, carbon fixation and gene silencing were widely affected, whereas 618 downregulated genes had an impact mainly on plant defence and carotenoid biosynthesis. Strikingly, all 11 highly responsive TFs carried abiotic stress response cis-regulatory elements, whereas five of them were better aligned with rice than with Arabidopsis gene homologues, suggesting that plant responses against viruses may predate divergence into monocots and dicots. Interestingly, tomato C2H2 family TFs, ZAT1-like and ZF2, may have distinct roles in plant defence due to opposite response patterns, similar to their Arabidopsis ZAT10 and ZAT12 homologues. These highly responsive TFs provide a basis to study in-depth molecular responses of the tomato-PepMV pathosystem, providing a perspective to better comprehend viral infections.

Identification and genetic diversity of grapevine virus L in Greece.

In this study, grapevine virus L (GVL) was identified for the first time in Greece through the application of high-throughput sequencing of total RNA from grapevine samples. Further investigation of the prevalence of GVL in Greek vineyards by RT-PCR revealed its presence in 5.5% (31/560) of the tested samples, which originated from six viticultural areas of the country. Comparative sequence analysis based on the CP gene revealed a high degree of genetic variability among GVL isolates, while phylogenetic analysis grouped the Greek isolates in three of the five phylogroups formed, with most of them being classified in phylogroup I.

Prevalence and Genetic Diversity of Viruses Associated with Rugose Wood Complex in Greek Vineyards.

Rugose wood is one of the most important disease syndromes of grapevine, and it has been associated with at least three viruses: grapevine rupestris stem pitting-associated virus (GRSPaV), grapevine virus A (GVA), and grapevine virus B (GVB). All three viruses show a worldwide distribution pattern, and their genetic composition has been the focus of extensive research in past years. Despite their first record in Greece almost 20 years ago, there is a lack of knowledge on the distribution and genetic variability of their populations in Greek vineyards. In this context, we investigated the distribution of GRSPaV, GVA, and GVB in rootstocks, self-rooted vines, and grafted grapevine cultivars originating from different geographic regions that represent important viticultural areas of Greece. Three new reverse transcription-PCR assays were developed for the reliable detection of GRSPaV, GVA, and GVB. Our results indicated that GVA is the most prevalent in Greek vineyards, followed by GRSPaV and GVB. However, virus incidence differed among self-rooted and grafted grapevine cultivars or rootstocks tested. Selected isolates from each virus were further molecularly characterized to determine their phylogenetic relationships. All three viruses exhibited high nucleotide diversity, which was depicted in the constructed phylogenetic trees. Isolates from Greece were placed in various phylogroups, reinforcing the scenario of multiple introductions of GVA, GVB, and GRSPaV in Greece and highlighting the effect of different transmission modes in the evolutionary course of the three viruses.

Molecular characterization of poleroviruses isolated from oilseed rape in Greece.

In 2018 virus-like symptoms, typical of polerovirus infection were observed in several oilseed rape crops in northern Greece. In order to identify the etiological agent of these symptoms a polerovirus-generic RT-PCR assay was applied. Sequencing of the amplicons revealed the presence of virus isolates genetically close to turnip yellows virus (TuYV). Further molecular characterization of the near complete genome of '1-2', 'Geo1', 'Geo7' and 'Geo15' isolates revealed that they share > 96% nt identity with various TuYV sequences. On the other hand, the fifth, characterized isolate from oilseed rape, termed '1-1', showed higher sequence similarity to brassica yellows virus (BrYV) regarding the 5' part of the complete coding sequence, whereas the 3' part was closely related to TuYV isolates. A recombination analysis using RDP indicated the presence of a putative breakpoint (nucleotide position 2964) in '1-1' genome and it is proposed that the virus isolate '1-1' might be an interspecies recombinant between BrYV and TuYV. To our knowledge, this is the first time that the complete coding sequences of Greek TuYV isolates have been determined and the first detection of a BrYV/TuYV recombinant isolate infecting oilseed rape in Greece.

The complete genome sequence of a divergent grapevine virus I isolate naturally infecting grapevine in Greece.

A significant number of new members of the genus Vitivirus have been identified recently, mainly due to the advent of high-throughput sequencing (HTS). Grapevine virus I (GVI), which was identified in New Zealand in 2018, is one of these viruses. RNAseq HTS analysis of a Greek grapevine (cv. Daphnia), revealed the presence of a GVI-like isolate (D2-1/19). Sequence analysis confirmed the classification of D2-1/19 as GVI. However, both sequence and phylogenetic data exhibited high levels of variability between D2-1/19 and the previously characterized GVI isolates. This study provides the full-length sequence of a divergent GVI isolate, adding knowledge to the limited information available about this recently identified virus.

Identification, molecular characterization and prevalence of a novel cytorhabdovirus infecting zucchini crops in Greece.

A new cytorhabdovirus was identified in zucchini (Cucurbita pepo) in Greece with the aid of high-throughput sequencing technology. The negative-sense, single-stranded genomic RNA of the new virus was determined and includes seven open reading frames in the order 3'-N-P-P3-P4-M-G-L-5' in the antigenomic orientation. Typical rhabdovirus-like particles were observed in infected leaf material. Comparative sequence analysis and phylogenetic reconstructions suggested that the described virus is a new member of the genus Cytorhabdovirus, and it was tentatively named cucurbit cytorhabdovirus 1 (CuCV1). To our knowledge CuCV1 is the first cytorhabdovirus infecting cucurbits in nature. Our surveys indicated that it occurs in a percentage of 36.7 % in zucchini crops in Greece.

Cucurbit chlorotic yellows virus: Insights Into Its Natural Host Range, Genetic Variability, and Transmission Parameters.

Cucurbit chlorotic yellows virus (CCYV) (genus Crinivirus, family Closteroviridae) is implicated in cucurbit yellows disease (CYV), causing typical interveinal yellowing symptoms in leaves, and is transmitted by Bemisia tabaci Mediterranean (MED) and Middle East-Asia Minor 1 (MEAM1). Due to its recent report in cucurbit crops in Greece, field surveys were conducted during 2011-2016 to determine the presence of the virus in symptomatic cucurbits and alternative hosts among arable weed species. Results indicated the restricted spread of the virus and identified 13 weed species as CCYV hosts for the first time. Sequence analysis of the RNA-dependent RNA polymerase (RNA1) coat and minor coat proteins (RNA2) revealed very low genetic diversity (<0.1%) among the Greek isolates. Transmission experiments were also conducted using B. tabaci MED with retention determined at four days, whereas transmission efficiency was positively correlated with the number of adults used, features linked to the virus semipersistent mode of transmission.

New poleroviruses associated with yellowing symptoms in different vegetable crops in Greece.

Four poleroviral isolates from Greece, two from lettuce, one from spinach and one from watermelon showing yellowing symptoms, were molecularly characterized by analyzing the sequence of a large part of the genome spanning from the 3'-terminal part of the RdRp to the end of the CP gene. The sequences were analyzed for their similarity and phylogenetic relationships to other members of the genus Polerovirus as well as for evidence of recombination events. The results revealed the existence of two putatively new viruses: one from lettuce and one from spinach, provisionally named "lettuce yellows virus" and "spinach yellows virus", respectively. Also, a new recombinant virus infecting lettuce, herein named "lettuce mild yellows virus", and a watermelon isolate of pepo aphid-borne yellows virus (PABYV) were identified. Our study highlights the existence of high genetic diversity within the genus Polerovirus, which could be associated with the emergence of new viral diseases in various crops worldwide.

Transmission of Tomato chlorosis virus (ToCV) by Bemisia tabaci Biotype Q and Evaluation of Four Weed Species as Viral Sources.

Tomato chlorosis virus (ToCV) is implicated in tomato yellows disease in many countries worldwide. It has a wide host range, including cultivated species as well as arable weeds, and it is transmitted in a semipersistent manner by at least five whitefly species or biotypes of the genera Trialeurodes and Bemisia. ToCV is not seed transmitted and more than 36 weed species have been recorded as natural reservoirs, acting as unique sources both for the virus and its vectors when susceptible crops are harvested. In this study, experiments were conducted to determine the transmission parameters of ToCV by biotype Q, the most abundant biotype of Bemisia tabaci in Greece. Results showed that biotype Q is an efficient vector of ToCV and it is able to retain the virus for at least 6 days. This vector was then used for the evaluation of four widespread weed species (Solanum nigrum, Sonchus oleraceus, Amaranthus retroflexus, and Chenopodium album) as ToCV sources through transmission experiments. Solanum nigrum was shown to be the most significant viral source among the tested weeds, followed by Sonchus oleraceus, A. retroflexus, and, lastly, C. album. Nevertheless, none of them was as efficient a ToCV source as tomato. This variation could be attributed to differences in virus concentration in each plant species or possible host preference by the whitefly vector.

First Report of Cucurbit chlorotic yellows virus in Cucumber, Melon, and Watermelon in Greece.

In 2011, an outbreak of a yellowing disease causing chlorosis and Interveinal chlorotic spots on lower leaves was observed in cucumber (Cucumis sativus) and melon (C. melo) plants in two greenhouses on the island of Rhodes, Greece. Similar symptoms were observed in 2012 in open field watermelon (Citrullus lanatus) plants in Rhodes and in November 2013 in a cucumber greenhouse in Tympaki, Crete. Disease incidence ranged from 10 to 40%. The observed symptoms were similar to those caused by whitefly transmitted criniviruses (family Closteroviridae) Cucurbit yellow stunting disorder virus (CYSDV) and Beet pseudo-yellows virus (BPYV), as well as Cucurbit chlorotic yellows virus (CCYV), a recently described crinivirus that infects cucurbits in Japan (4) and by the aphid transmitted polerovirus (family Luteoviridae) Cucurbit aphid-borne yellows virus (CABYV). Dense populations of whiteflies were present in all the affected crops. Leaf samples from cucumber (10 from Rhodes and 10 from Crete), melon (10), and watermelon (10) were collected and tested for the presence of the above viruses. Total RNA was extracted from the samples (2) and detection of BPYV, CYSDV, and CABYV was done as previously described (1,3) whereas detection of CCYV was conducted by herein developed two-step RT-PCR assays. Two new pairs of primers, 'CC-HSP-up' (5'-GAAGAGATGGGTTGGTGTAGATAAA-3')/'CC-HSP-do' (5'-CACACCGATTTCATAAACATCCTTT-3') and 'CC-RdRp-up' (5'-CCTAATATTGGAGCTTATGAGTACA-3')/'CC-RdRp-do' (5'-CATACACTTTAAACACAACCCC-3') were designed based on GenBank deposited sequences of CCYV for the amplification of two regions partially covering the heat shock protein 70 homologue (HSP70h) (226 bp) and the RNA dependent RNA polymerase (RdRp) genes (709 bp). Interestingly, CCYV was detected in all samples tested, while CYSDV was detected in 18 cucumbers (10 from Rhodes and 8 from Crete), 1 melon, and 3 watermelon plants. Neither BPYV nor CABYV were detected. In order to verify the presence of CCYV, the partial HSP70h and RdRp regions of a cucumber isolate from Crete were directly sequenced using the primers 'CC-HSP-up'/'CC-HSP-do' and 'CC-RdRp-up'/'CC-RdRp-do'. BLAST analysis of the obtained sequences (HG939521 and 22) showed 99% and 100% identities with the HSP70h and RdRp of cucumber CCYV isolates from Lebanon, respectively (KC990511 and 22). Also, the partial HSP70h sequence of a watermelon CCYV isolate from Rhodes showed 99% identity with the cucumber isolate from Crete. Whitefly transmission of CCYV was also carried out by using an infected cucumber from Crete as virus source. Four groups of 30 whitefly adults of Bemisia tabaci biotype Q were given an acquisition and inoculation access time of 48 and 72 h, respectively. Each whitefly group was transferred to a healthy cucumber plant (hybrid Galeon). Two weeks post inoculation, the plants, which have already been showing mild interveinal chlorosis, were tested for virus presence by RT-PCR. CCYV was successfully transmitted in three of four inoculated cucumbers, which was further confirmed by sequencing. In Greece, cucurbit yellowing disease has occurred since the 1990s, with CYSDV, BPYV, and CABYV as causal agents. To our knowledge, this is the first report of CCYV infecting cucurbits in Greece; therefore, our finding supports the notion that the virus is spreading in the Mediterranean basin and is an important pathogen in cucurbit crops. References: (1) I. N. Boubourakas et al. Plant Pathol. 55:276, 2006. (2) E. Chatzinasiou et al. J. Virol. Methods 169:305, 2010. (3) L. Lotos et al. J. Virol. Methods 198:1, 2014. (4) M. Okuda et al. Phytopathology 100:560, 2010.

Epidemiology and genetic diversity of criniviruses associated with tomato yellows disease in Greece.

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.

Detection and quantification of infectious avian influenza A (H5N1) virus in environmental water by using real-time reverse transcription-PCR.

Routes of avian influenza virus (AIV) dispersal among aquatic birds involve direct (bird-to-bird) and indirect (waterborne) transmission. The environmental persistence of H5N1 virus in natural water reservoirs can be assessed by isolation of virus in embryonated chicken eggs. Here we describe development and evaluation of a real-time quantitative reverse transcription (RT)-PCR (qRT-PCR) method for detection of H5N1 AIV in environmental water. This method is based on adsorption of virus particles to formalin-fixed erythrocytes, followed by qRT-PCR detection. The numbers of hemagglutinin RNA copies from H5N1 highly pathogenic AIV particles adsorbed to erythrocytes detected correlated highly with the infectious doses of the virus that were determined for three different types of artificially inoculated environmental water over a 17-day incubation period. The advantages of this method include detection and quantification of infectious H5N1 AIVs with a high level of sensitivity, a wide dynamic range, and reproducibility, as well as increased biosecurity. The lowest concentration of H5N1 virus that could be reproducibly detected was 0.91 50% egg infective dose per ml. In addition, a virus with high virion stability (Tobacco mosaic virus) was used as an internal control to accurately monitor the efficiency of RNA purification, cDNA synthesis, and PCR amplification for each individual sample. This detection system could be useful for rapid high-throughput monitoring for the presence of H5N1 AIVs in environmental water and in studies designed to explore the viability and epidemiology of these viruses in different waterfowl ecosystems. The proposed method may also be adapted for detection of other AIVs and for assessment of their prevalence and distribution in environmental reservoirs.

Demarcation of ilarviruses based on the phylogeny of RNA2-encoded RdRp and a generic ramped annealing RT-PCR.

In this study, a generic ramped-annealing (RAN) nested RT-PCR was developed, allowing the simultaneous detection and fast characterization of ilarviruses. The method involves a one-step RT-PCR in which a pair of degenerate primers amplifies a 381-bp part of the polymerase gene (RNA2), followed by a nested PCR amplification that increases detection sensitivity. The sensitivity and detection range of the method were further increased by applying a ramped annealing thermocycling step both in the first RT-PCR and in the subsequent nested PCR. The 371-bp nested amplicons can be sequenced directly, without cloning, to obtain initial sequence information on ilarvirus genomes, or can undergo a restriction enzyme analysis for rapid identification of already known virus species. Phylogenetic relationships among different members of the family Bromoviridae were inferred with maximum likelihood and Bayesian analysis, using published homologous partial amino acid sequences corresponding to the nested amplicon and also to a longer residue data set (432-453 aa) comprising all possible positions of homology among the RNA2-encoded polymerases of members of the family Bromoviridae. The implications of these analyses on the taxonomy of ilarviruses are discussed. The specific partial polymerase sequence, corresponding to the polymerase core palm structure (motifs A-D), was verified as phylogenetically informative and can be used to separate ilarviruses from other members of the family Bromoviridae, providing initial information for ilarvirus species characterization. However, the phylogenetic signal of this region is not reliable for inferring relationships among distantly related ilarviruses.