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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.

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.

Grapevine yellow speckle viroid-2 is a small, circular, single-stranded RNA molecule that is classified in the genus Apscaviroid (family Pospiviroidae). Grapevine yellow speckle viroid-2 (GYSVd-2) has been identified in many grapevine-producing countries such as India, Australia, China, Thailand (Singhal et al. 2019; Tangkanchanapas et al. 2017), Turkey, and Italy (Gambino et al. 2014). Until now, no information regarding its presence and spread in Greek vineyards was available. For this purpose, a random survey was conducted, and 327 samples of leaves and stems were collected from a large range of cultivars from Crete, Attiki, Peloponnese, Northern Greece, and Dodecanese. Total RNA was extracted according to the protocol of Maliogka et al. (2015), and reverse transcription PCR (RT-PCR) was performed using a new primer pair, GYSVd-2 up (5′-GACCTGCAGAGAAAAGAAGAAGG-3′)/GYSVd-2 do (5′-GCTCGACTAGCGGAGGC-3′). GYSVd-2 was frequently identified in the tested samples (39/327). The full viroid genomes of two isolates from Cretan vines (cv. Soultanina) and two from Peloponnese (cv. Superior) were amplified and sequenced using two different pairs of primers: GYSVd-2up 2 (5′-CCTAAGATGCCTCCGCTAGT-3′)/GYSVd-2do 2 (5′-AGACAGGCCCGCGTTTCG-3′) and GYSVd-2up3 (5′-AGAAGGGCCCAGAGGGGAAT-3′)/GYSVd-2do3 (5′-TCTTTTCTCTGCAGGTCCGC-3′). Sanger sequencing of both amplicons was performed in order to confirm the primer annealing sites. The 363-bp amplified genomes of the Cretan isolates (deposited in ENA under accession nos. LR735992 and LR735993) revealed high identity with GYSVd-2 isolates deposited in GenBank (98.63 to 100% nt identity) and 100% nt identity among them, whereas the isolates from Peloponnese (accession nos. LR735995 and LR735996) revealed 98.9 to 100% nt identity to deposited viroid isolates. At the same time the viroid was detected during a high-throughput sequencing analysis of rRNA depleted total RNA from a grapevine (cv. Mavro Nemeas) from Peloponnese. Sequencing was conducted on an Illumina NovaSeq6000 platform yielding ∼60 million 100-bp paired-end reads. After extraction of the Vitis vinifera genome, the de novo assembly of the reads using SPAdes produced 20,365 contigs. The 1,411 contigs exhibiting a size over 240 nt were screened against the nt/nr databases using BLAST. Among the identified viral sequences, the 363-nt-long genome of GYSVd-2 (LR735994) was retrieved, showing 100% nt identity to the cultivar Superior isolate from Peloponnese as well as to isolates from Croatia, Pakistan, and China (MF979530, KY978405, and FJ490172, respectively). The presence of the viroid in this sample was confirmed with the aforementioned RT-PCRs. To our knowledge, this is the first report of GYSVd-2 in Greece, which is expanding our knowledge on its worldwide distribution. Our initial data indicate that the viroid is dispersed in several regions of Greece, and it shows rather low genetic variability. Additional work is needed to elucidate its spread and impact on Greek grapevines.

Avocado (Persea americana) is an important fruit crop for the island of Crete, in which almost the total yield of the fruit in Greece is being produced. The total cultivation area is estimated at more than 400 ha (FAOSTAT 2015). During the growing season of 2016 fruits exhibiting depressed greenish yellow-white craters, which are typical symptoms of avocado sunblotch disease, were spotted on a single tree in an orchard located in the province of Chania, Crete. No symptoms were observed on the leaves or shoots of the tree nor on any other tree in the orchard. The causal agent of sunblotch disease is avocado sunblotch viroid (ASBVd-family Avsunviroidae). In order to determine if the viroid was present, total RNA preparations from leaves, fruit skin and pulp from the symptomatic tree were extracted (Maliogka et al. 2015) and tested by a two-step RT-PCR (Schnell et al. 1997), which amplifies the cDNA of the complete ASBVd genome. Leaves from nine non-symptomatic trees, from the same field, were also analyzed. The RT-PCR performed on all the tissues of the symptomatic sample yielded the expected unit-length amplicon, as well as the dimeric and polymeric forms of the viroid, whereas all the non-symptomatic samples were found negative. The unit-length amplicon was directly sequenced, whereas the dimeric amplicon was excised from the gel, purified and cloned into a pGEM®-T Easy vector (Promega) and subsequently sequenced (VBC-Biotech). The whole genome sequence of ASBVd (249 nucleotides) was determined from the monomeric RT-PCR product and from two dimeric clones and was deposited in European Nucleotide Archive (ENA). Two different variants were identified from the symptomatic fruit, AvoCr0-A and B (Acc. No. LT966069 and LT966070, respectively). BLASTn results showed that the Greek variants of ASBVd (AvoCr0-A and B) are 95-99% identical to the already characterized isolates of ASBVd. The presence of the viroid in the symptomatic tree was also confirmed by northern blot analysis (Torchetti et al. 2012). For this aim, total RNAs were separated by 5% PAGE electrophoresis in denaturing conditions, electro-transferred to a nylon membrane and hybridized with a DIG-labeled full-length RNA probe specific for the plus strand of ASBVd (provided by Dr. Ricardo Flores, UPV-CSIC, Spain). Circular and linear forms of ASBVd RNAs co-migrating with those of the positive control (also provided by Dr. R. Flores) were observed. The presence of additional infected trees in the same field is not excluded, but further investigation is needed to obtain conclusive data in this respect. To our knowledge, this is the first report of ASBVd in Greece. Given the importance of the sunblotch disease, future studies should be performed to determine the prevalence and the significance of this viroid in Greek orchards.

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.

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.

Grapevine virus H(GVH) is a member of the genusVitivirusin the familyBetaflexiviridae(subfamilyTrivirinae, orderTymovirales) that infects grapevine (Candresse etal. 2018). GVH was first identified in a symptomless grapevine of an unknown cultivar from Portugal in 2018 (Candresse etal. 2018), and since then the virus has been reported only from California (Diaz-Lara etal. 2019). Several vitiviruses have been detected in Greek vineyards (Avgelis and Rumbos 2000;Dovas and Katis 2003a,b;Lotos etal. 2020;Panailidou etal. 2019), 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 ofWhite etal. (2008)and subjected to rRNA depletion, library construction (TruSeq Stranded Total RNA kit), and high-throughput sequencing (HTS) in a NovaSeq6000 platform (Illumina) 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 was 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 United States (MN716768.1). 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 reverse transcription polymerase chain reaction, 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 the United States, respectively (NC_040545.1 and MN716768.1), 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.