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.

Characterization of Pepper leafroll chlorosis virus, a New Polerovirus Causing Yellowing Disease of Bell Pepper in Saudi Arabia.

During the growing seasons of 2014 through 2016, a total of 336 leaf samples from bell pepper (showing leafroll and interveinal yellowing) and arable weeds were collected from Riyadh region, Saudi Arabia. The use of a polerovirus generic reverse transcription (RT)-PCR assay confirmed their presence in the bell pepper samples. Sequencing of the generic amplicon revealed high similarity (87.6 to 98.1% in nt) with four poleroviruses; Tobacco vein distorting virus, Pepper vein yellows virus, Pepper yellows virus, and Pepper yellow leaf curl virus. To further characterize one of these isolates (105D), a larger part of the genome (∼1,300 nt) spanning approximately from the 3' end of ORF2 to the middle of ORF3, was amplified and sequenced. Blasting the resulting sequence revealed the low amino acid and nucleotide identity percentages in the coat protein and movement protein partial genes with viruses deposited in GenBank. Next-generation sequence was used to acquire a larger part of the genome, which resulted in the reconstruction of isolate 105D's partial genome (5,496 nt). Sequence similarity analysis revealed the presence of a divergent polerovirus isolate belonging to a new species that was tentatively named Pepper leafroll chlorosis virus (PeLRCV). Using a specific RT-PCR assay for this isolate confirmed the presence of this new viral species in the symptomatic peppers. Aphid transmission experiments showed that PeLRCV is vectored by Aphis gossypii and that it can infect at least five out of the 15 different plants species tested. Based on our findings, PeLRCV is a new member of genus Polerovirus in the family Luteoviridae.

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.

Complete genome analysis and immunodetection of a member of a novel virus species belonging to the genus Ampelovirus.

A new grapevine leafroll-associated virus isolate (GLRaV-Pr) from Greek grapevines was recently reported. This virus, along with the genetically related GLRaV-4, -5, -6 and -9, form a separate diverse lineage within the genus Ampelovirus. In this paper, the complete nucleotide sequence of GLRaV-Pr was determined, making it the first fully sequenced virus of this lineage. Its genome is 13,696 nt long and contains seven open reading frames, which potentially encode a 253-kDa polyprotein containing papain-like protease, methyltransferase, AlkB and helicase domains, a 58.2-kDa RNA-dependent RNA polymerase, a 5.2-kDa hydrophobic protein, a 58.5-kDa heat shock 70 protein homologue, a 60-kDa protein, a 30-kDa coat protein (CP) and a 23-kDa protein. A virus-specific antibody was raised against the recombinant CP of GLRaV-Pr and was applied in western blot analysis. The genomic, serological and phylogenetic data reported here confirm that GLRaV-Pr is a member of a distinct Ampelovirus species. Comparisons of GLRaV-Pr with the only available genetically related, fully sequenced virus, PMWaV-1, PBNSPaV and the partially sequenced GLRaV-9 revealed that this lineage, including GLRaV-4, -5, -6, -9 and -De, exhibits a high uniformity of genome organization and includes the smallest and simplest viruses within the family Closteroviridae.

First Report of Botrytis cinerea Causing Gray Mold of Pomegranate (Punica granatum) in Greece.

Pomegranate is rapidly increasing in production in Greece. During August of 2008 in the region of Larisa (central Greece), preharvest fruit rot was observed on pomegranate (cv. Kapmaditika) that caused losses estimated at 10%. Symptoms first appeared as small spots on the fruits that later increased in size and developed into expanded, dark brown lesions. Internally, tissues were soft and brown with gray mycelia and conidiophores observed. Affected fruits decayed completely during 2 months of storage (5 to 6°C), causing yield losses of up to 20%. To isolate the casual agent, conidia and conidiophores were scraped aseptically from the internal tissues, suspended in sterile water, and streaked onto the surface of potato dextrose agar (PDA). Single hyphal tips were transferred to PDA, and the isolated fungus was identified as Botrytis cinerea Pers.:Fr. on the basis of morphological characteristics (2). B. cinerea was consistently isolated from symptomatic tissues. Colonies of B. cinerea on PDA were at first colorless and became gray to brown with the development of lemon-shaped conidia (average 7.5 × 9 µm). Sclerotia were black and varied in size (1.4 to 4.5 × 1.5 to 2.7 mm) and shape (2). Pathogenicity of the isolated fungus was tested by wound inoculating five mature pomegranate fruits (cv. Kampaditika) after surface sterilization with 5% sodium hypochlorite. Plugs of the fungus (5 mm in diameter) obtained from the colony margins were transferred onto a 3- × 3-mm wound on the surface of sterilized fruit. Sterile PDA plugs were used to inoculate five control pomegranate fruits. Fruit were incubated at 22°C and 80% relative humidity in the dark. Extensive decay, similar to that observed on diseased fruits in the field, was observed on inoculated fruits 7 days after inoculation, whereas control fruits showed no decay. The pathogen was reisolated from internal rotten tissues of inoculated fruit, but not from the noninoculated control fruits. Fruit rot of pomegranate caused by B. cinerea has been reported previously in the United States (1) and China (3). However, to our knowledge, this is the first report of B. cinerea causing gray mold of pomegranate in Greece. References: (1) A. M. French. California Plant Disease Host Index. Calif. Dept. Food Agric., Sacramento, 1989. (2) W. B. Hewitt. Compendium of Grape Diseases. American Phytopathological Society, 1994. (3) Z. Zhang. Flora Fungorum Sinicorum 26:277, 2006.

First Report of Penicillium glabrum Causing Fruit Rot of Pomegranate (Punica granatum) in Greece.

During September and October of 2008 in the region of Larisa (central Greece), postharvest fruit rot was observed on pomegranate (cv. Kapmaditika), which is rapidly increasing in production in Greece, causing losses of 10 to 20% after 2 months of cold storage (5 to 6°C). Infected fruits showed green conidiophores in the calyx area, while internal symptoms consisted of soft, brown tissue that became covered with green mycelium and conidiophores. To isolate the casual agent, conidia and conidiophores were scraped aseptically from the internal fruit rot, suspended in sterile water, and streaked onto potato dextrose agar (PDA). Single hyphal tips were then transferred to new PDA plates. A fungus consistently isolated from the infected tissues was identified as Penicillium glabrum (Wehmer) Westling on the basis of morphological criteria, with conidiophores smooth or finely roughened and conidia in compact columns, glubose to subglubose, approximately 3.0 µm, with walls somewhat echinulate (1). The identification was confirmed by sequencing the internal transcribed spacer (ITS) region spanning ITS1, 5.8S, and ITS2 of the ribosomal DNA (2). The nucleotide sequence was submitted to GenBank (Accession No. FN313540). The pathogenicity of the isolated fungus was tested on five mature pomegranate fruit (cv. Kampaditika) after being surface sterilized with 5% sodium hypochlorite. A plug (5 mm in diameter) obtained from the margins of a P. glabrum colony was transferred to wounds (3 × 3 mm) made with a scalpel in the surface of fruit. Fruit inoculated with sterile PDA plugs served as controls. Fruit were incubated at 22°C and 80% relative humidity in the dark. Extensive decay, similar to that observed on diseased fruit in the field, was observed on the inoculated fruit 7 days after inoculation, whereas control fruit showed no decay. The pathogen was reisolated from inoculated fruit but not from the noninoculated fruit. To our knowledge, this is the first report of P. glabrum causing postharvest fruit rot of pomegranates in Greece. References: (1) C. Thom and K. B. Raper. Page 176 in: A Manual of the Penicillia. Williams and Wilkins, Baltimore, 1949. (2) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.