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.

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.

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.

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 Tomato chlorosis virus (ToCV) in Tomato Crops in Saudi Arabia.

During January 2014, open field and greenhouse tomato (Solanum lycopersicum L.) crops in the peripheral areas of Riyadh region (Al-Aflaj, Al-Kharj, Al-Waseel, and Al-Dalam), Saudi Arabia, were surveyed. In all surveyed tomato crops, yellowing symptoms were observed on the lower leaves, possibly infected by a whitefly transmitted crinivirus (family Closteroviridae) such as Tomato chlorosis virus (ToCV) and/or Tomato infectious chlorosis virus (TICV). Dense population of whiteflies (Bemisia tabaci G.) were present in all affected plants. Incidence of the yellowing disease varied between four greenhouses and three open field tomato crops, but in the majority of the tomato crops surveyed, symptoms typical of Begomovirus infection such as severe stunting, degeneration, upward cupping, distortion and interveinal yellowing of upper leaves, and flower abortion were also observed. Tomato yellow leaf curl virus (TYLCV) is endemic in Saudi Arabia causing severe crop losses (1). Twenty-six leaf samples from 24 symptomatic and two asymptomatic plants from four fields (three greenhouses and one open field crop) were collected and were processed in the lab at King Saud University. Whitefly transmission on tomato indicator plants was carried out using B. tabaci to fulfill Koch's postulates. Two hundred virus-free B. tabaci adults were confined to one of the collected symptomatic tomato sample singly infected with ToCV for a 48-h acquisition access period, followed by a 48-h inoculation access period on five healthy tomato plants Hybrid Super Strain B, using 40 whiteflies per plant. Crinivirus detection following transmission was conducted by RT-PCR. Total RNA was extracted from 26 collected leaf samples using the Total RNA Purification Kit and analyzed by SCRIPT One-Step RT-PCR Kit (Jena Bioscience). First, the degenerate primers HS-11/HS12 were used for amplification of a 587-bp fragment of the HSP70 gene of ToCV and TICV (3). Second, the RT-PCR product was subjected to a nested PCR using specific primers TIC-3/TIC-4 and TOC-5/TOC-6, for the detection of both TICV and ToCV, respectively (2). Finally, degenerate primers (AV494/AC1048) were used for detection of begomoviruses (4). No fragment was amplified by TIC-3/TIC-4 primer whereas TOC-5/TOC-6 amplified a size of 463 bp in all 24 symptomatic tested samples, including one mixed infection with TYLCV detected by AV494/AC1048. Asymptomatic samples did not produce any amplicon regarding TICV, ToCV, and Begomovirus detection. The amplicons of four positive fragments, each from one field, were further sequenced in both directions and all obtained sequences (KJ433488, KJ433489, KJ433490, and KJ433491) analyzed with BLAST and revealed 99% identity with the most closely deposited sequences in NCBI from Japan (AB513442) and Brazil (JQ952601). In the transmission tests, ToCV was detected to all tomato indicator plants which revealed yellowing symptoms 6 weeks post inoculation, whereas no transmission was obtained when non-viruliferous whitefly adults fed on two asymptomatic tomato leaves. To our knowledge, this is the first report of ToCV infecting tomato crops in Saudi Arabia. Further studies are being carried out to study epidemiology and genetic diversity of this virus associated with yellowing diseases of tomato in different regions of Saudi Arabia. This finding is important for the tomato crops and possibly other virus hosts as may cause serious epidemics and crop losses. References: (1) A. M. Ajlan et al. Arab J. Biotech. 10:179, 2007. (3) C. I. Dovas et al. Plant Dis. 86:1345, 2002. (2) J. Navas-Castillo et al. Plant Dis. 84:835, 2000. (4) S. D. Whyatt and J. K. Brown. Phytopathology 86:1288, 1996.

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, 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.

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.