Greek Vineyards Research Articles

Improved detection, mealybug transmission and prevalence of grapevine Roditis leaf discoloration-associated virus in Greek vineyards

AbstractGrapevine Roditis leaf discoloration-associated virus (GRLDaV) is a member of the species Badnavirus decoloratiovitis in the genus Badnavirus that was recently identified in grapevine using high throughput sequencing. In this study, a large-scale survey was conducted to monitor the prevalence of the virus in major viticultural areas of Greece. A total of 970 samples were collected from vines and molecularly analysed using PCR assays targeting two regions of the virus genome. GRLDaV was detected in 8 geographic areas of Greece, showing high frequency in southern and central Greece and an overall prevalence of 17.1%. GRLDaV was identified mainly in autochthonous grapevine varieties, and it showed higher frequency in the self-rooted ones, especially in the geographic region of the Cyclades islands. In transmission tests conducted using Planococcus ficus (Signoret) and 48 h acquisition access period (AAP) and inoculation access period (IAP), the virus was transmitted with an efficiency of 14.3% (3/21 grapevines). Finally, electron microscopy revealed the bacilliform morphology of GRLDaV virions, thus further confirming its episomal infection.

Prevalence and molecular characterization of grapevine virus E, F and I populations in Greek vineyards

In this study, RT-PCR assays were developed and applied to investigate the prevalence of grapevine virus E (GVE), grapevine virus F (GVF) and grapevine virus I (GVI) in Greek vineyards. Grapevine samples from different viticultural areas of Greece were tested and the presence of all three vitiviruses was revealed. These viruses were mainly detected in grafted indigenous grapevine cultivars, with GVF being the most prevalent (24.7%, 200/809) in contrast to GVI (2.2%, 12/554) and GVE (1.6%, 12/752). To further study their intraspecies genetic variability and the phylogenetic relationships of their populations, sequence similarity analyses of the replicase and the coat protein gene segments of each virus, as well as from a fragment of the movement protein gene of GVI, were performed. Results revealed genetic variability in all three virus populations and high molecular diversity between Greek isolates of GVE and GVF, in contrast to Greek GVI isolates, which showed high homogeneity. Overall, our study advanced the current knowledge on grapevine-infecting vitiviruses and highlighted that special attention should be given to the widely spread GVF and its impact on grapevine should be further investigated.

Resistance of Black Aspergilli Species from Grape Vineyards to SDHI, QoI, DMI, and Phenylpyrrole Fungicides.

Fungicide applications constitute a management practice that reduces the size of fungal populations and by acting as a genetic drift factor, may affect pathogen evolution. In a previous study, we showed that the farming system influenced the population structure of the Aspergillus section Nigri species in Greek vineyards. The current study aimed to test the hypothesis that the differences in the population structure may be associated with the selection of fungicide-resistant strains within the black aspergilli populations. To achieve this, we determined the sensitivity of 102, 151, 19, and 22 for the A. uvarum, A. tubingensis, A. niger, and A. carbonarious isolates, respectively, originating either from conventionally-treated or organic vineyards to the fungicides fluxapyroxad-SDHIs, pyraclostrobin-QoIs, tebuconazole-DMIs, and fludioxonil-phenylpyrroles. The results showed widespread resistance to all four fungicides tested in the A. uvarum isolates originating mostly from conventional vineyards. In contrast, all the A. tubingensis isolates tested were sensitive to pyraclostrobin, while moderate frequencies of only lowly resistant isolates were identified for tebuconazole, fludioxonil, and fluxapyroxad. Sequencing analysis of the corresponding fungicide target encoding genes revealed the presence of H270Y, H65Q/S66P, and G143A mutations in the sdhB, sdhD, and cytb genes of A. uvarum resistant isolates, respectively. No mutations in the Cyp51A and Cyp51B genes were detected in either the A. uvarum or A. tubingensis isolates exhibiting high or low resistance levels to DMIs, suggesting that other resistance mechanisms are responsible for the observed phenotype. Our results support the initial hypothesis for the contribution of fungicide resistance in the black aspergilli population structure in conventional and organic vineyards, while this is the first report of A. uvarum resistance to SDHIs and the first documentation of H270Y or H65Q/S66P mutations in sdhB, sdhD, and of the G143A mutation in the cytb gene of this fungal species.

Comparative Assessment of Environmental/Energy Performance under Conventional Labor and Collaborative Robot Scenarios in Greek Viticulture

The viticultural sector is facing a significant maturation phase, dealing with environmental challenges to reduce agrochemical application and energy consumption, while labor shortages are increasing throughout Europe and beyond. Autonomous collaborative robots are an emerging technology and an alternative to the scarcity of human labor in agriculture. Additionally, collaborative robots could provide sustainable solutions to the growing energy demand of the sector due to their skillful precision and continuous labor. This study presents an impact assessment regarding energy consumption and greenhouse gas emissions of collaborative robots in four Greek vineyards implementing a life cycle assessment approach. Eight scenarios were developed in order to assess the annual production of four Vitis vinifera L. cultivars, namely, Asyrtiko, Cabernet Sauvignon, Merlot, and Tempranillo, integrating data from two wineries for 3 consecutive years. For each conventional cultivation scenario, an alternative was developed, substituting conventional viticultural practices with collaborative robots. The results showed that collaborative robots’ scenarios could achieve a positive environmental and energy impact compared with conventional strategies. The major reason for lower impacts is fossil fuel consumption and the efficiency of the selected robots, though there are limitations regarding their functionality, lifetime, and production. The alternative scenarios have varying energy demand and environmental impact, potentially impacting agrochemical usage and requiring new policy adjustments, leading to increased complexity and potential controversy in farm management. In this context, this study shows the benefits of collaborative robots intended to replace conventional practices in a number of viticultural operations in order to cope with climate change impacts and excessive energy consumption.

Conventional vs. organic vineyards: Black Aspergilli population structure, mycotoxigenic capacity and mycotoxin contamination assessment in wines, using a new Q-TOF MS-MS detection method

Aspergillus bunch rot, caused by several mycotoxigenic species of Black Aspergilli section Nigri is one of the most severe diseases of grapevine, while mycotoxin contaminated products may pose a risk to the consumers health. During a 2-year (2018–2019) survey, the impact of conventional and organic farming systems on Black Aspergilli population structure in Greek vineyards was thoroughly investigated. In total, 300 isolates of Aspergillus spp. were selected and identified by amplicon sequencing of the ITS-region, β-tubulin and calmodulin genes. Four different Aspergillus section Nigri species (A. tubingensis, A. uvarum, A. carbonarius and A. niger) were identified as the casual agents of the disease. During both sampling years, higher frequencies of A. tubingensis and A. uvarum were found in the organic and conventional vineyards, respectively. In vitro production of ochratoxin A (OTA) and fumonisin B2, B3 and B4 (FB2, FB3, and FB4) was evaluated in two selective media. The analysis revealed a low frequency of mycotoxigenic isolates, while, most of these isolates originated from conventional vineyards. As expected, A. carbonarious was identified as the main OTA producer (0.01–9.2 μg/g), whereas A. niger was the leading producer of FBs. In total, 74 wine samples originating from either organic or conventional vineyards of the same region, were analyzed using a new Quadrupole Time-of-Flight Mass Spectrometry (QTOF MS-MS) analytical method developed to detect and quantify Alternariol (AOH), Alternariol monomethyl ether (AME), and Citrinin (CIT) in addition to the aforementioned Aspergillus mycotoxins (OTA, FB1, FB2 FB3), using a modified QuEChERS extraction protocol. Interestingly, a low frequency of mycotoxin-contaminated wines was detected. At the same time, fumonisins (FBs) were identified as the main mycotoxins in Greek wines. This is the first study deciphering the impact of conventional and organic farming systems on Aspergillus section Nigri species in Greek vineyards, suggesting that farming system may affect the species composition within the vineyard. Furthermore, evidence was provided for a higher risk of selection of Aspergillus strains with higher mycotoxigenic capacity in conventional vineyards, associated with higher risk of mycotoxin contamination in wines originating from these vineyards.

Genetic diversity of Greek grapevine (Vitis viniferaL.) cultivars using ampelographic and microsatellite markers

AbstractGrapevine (Vitis viniferaL.) is a major worldwide crop of high economic importance, tightly interwoven with the traditions and the culture of many civilizations. The Greek vineyard is one of the oldest in the world composed of an ample number of highly diverse indigenous landraces. However, over the last decades the local cultivated grapevine germplasm has undergone a drastic reduction of diversity, due to the established market preferences for international varieties. In the current work a combined approach involving both, ampelographic traits and microsatellite markers has been undertaken, to study the genetic diversity within and among 96 grapevine genotypes belonging to 36V. viniferasubsp.viniferacultivars, predominantly representing autochthonous Greek landraces. Results revealed high genetic diversity for the Greek cultivars yielding a mean number of alleles per locus 14.69 and mean polymorphic information content 0.848. Hierarchical cluster analysis, employing both, ampelographic and microsatellite data, showed a clear distinction based on the origin of the germplasm; Anatolian versus Mediterranean. Principal component analysis, based on the most informative ampelographic traits, coupled to the results from genetic structure analysis further corroborated the proposal of germplasm differentiation on the basis of geographic origin. This information can be further utilized for the reconstitution of the Greek vineyard and can significantly contribute towards a rational conservation and utilization strategy for breeding or for direct cultivation of the Greek indigenous grapevine germplasm.

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.

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 (Diaz‑Lara 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.

Use of UAV-borne multispectral data and vegetation indices for discriminating and mapping three indigenous vine varieties of the Greek Vineyard

Use of UAV-borne multispectral data and vegetation indices for discriminating and mapping three indigenous vine varieties of the Greek Vineyard

First Report of Grapevine Yellow Speckle Viroid-2 in Grapevine in Greece

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.

Grapevine Phenological Quantitative Trait SSR Genotyping Using High-Throughput HRM-PCR Analysis

Discrimination among grapevine varieties based on quantitative traits, such as flowering, veraison and ripening dates is crucial for variety selection in the context of climate change and in breeding programs. These traits are under complex genetic control for which 6 linked SSR loci (VVS2, VVIn16, VMC7G3, VrZAG29, VMC5G7, and VVIB23) have been identified. Using these markers in HRM-PCR analysis, we assessed genetic diversity among a large collection of 192 grapevine varieties. The grapevine germplasm used encompasses the majority of Greek vineyard with 181 varieties, 3 prominent foreign varieties and 11 varieties of Palestinian origin. The SSR markers used were highly polymorphic, displaying unique melting curves for unusually higher number of samples than generally observed in SSR analysis. This prompted us to examine sequence composition for selected samples and found that variation present as SNPs in the flanking sequences of SSR motifs was responsible for the observed polymorphism. Hence, HRM-PCR proved to be a tool of higher analytical power to distinguish genotypes surpassing the discrimination power of conventional gel-based SSR analysis. The study provides a better understanding of genetic variation of SSR marker loci associated to phenological traits in grapevine varieties, signifying an analytical methodology that may be of higher discrimination power in detection of polymorphism for utilization in breeding programs.

Comparative study of qualitative and quantitative characters of grape cultivar 'Mavrodafni' (Vitis vinifera L.) and 'Renio' grown in different regions of the Protected Designation of Origin Mavrodafni Patras

'Mavrodafni' (Vitis vinifera L.) is considered one of the oldest grapevine cultivars indigenous to the Greek vineyards, with western Peloponnese being its primary centre of cultivation. 'Renio' is considered to be either a biotype of 'Mavrodafni' or an altogether different cultivar. Both 'Mavrodafni' and 'Renio' can be found in the vineyards of the areas of cultivation, since 'Renio' is considered to be more productive compared to 'Mavrodafni' and, for this reason, it has gradually replaced 'Mavrodafni' from cultivation over the course of time. 'Mavrodafni' and 'Renio' contain appreciable amounts of quality characters of grape and must in terms of total polyphenol and total anthocyanins content in berry skins, depending on the different regions where they are cultivated and they would be worthy of further study and use for the production of different types of wines.

Differentiation of Greek grapevine cultivars (Vitis vinifera L.) based on the combination of ampelographic description and microsatellite markers

Greek vineyards are characterized by a great number of indigenous grapevine cultivars as well as grapevine cultivars of eastern origin. Due to their age-old lineage and cultivation in Greece, most of those cultivars exhibit an even greater number of biotypes and mutations which frequently lead to an improper use of synonyms and homonyms. The aim of this study was the identification and discrimination of twelve grapevine cultivars cultivated in Greece by means of the seventy-four OIV descriptors, followed by the molecular analysis of five microsatellite markers on each grapevine biotype and cultivar during the years 2015, 2016, and 2017, on the basis of a list of ampelographic descriptors developed by the International Organization of Vine and Wine (OIV in OIV descriptor list for grape varieties and Vitis species, 2nd edn, Organisation Internationale de la Vigne et du Vin, 2009. http://oiv.int/en/technical-standards-and-documents/description-of-grape-varieties), followed by the molecular analysis of five microsatellite markers. The studied grapevine cultivars were clustered according to their origin; grapevine cultivars of eastern origin were placed within the same cluster of the dendrogram, while the high degree of genetic similarity of grapevine cultivars ‘Avgoulato’, ‘Korithi lefko’, ‘Kolokythas’ and ‘Baresana’ bear to one another corroborates the hypothesis that all of them were formed by mutations from the same single cultivar. With regard to the indigenous grapevine cultivars, ‘Zalovitiko’ proved to be a synonym of ‘Xinomavro’, while the low degree of genetic similarity between grape cultivars ‘Korithi mavro’ and ‘Korithi lefko’ suggests that they are different and do not constitute colour mutations, as it had been believed so far. In most cases, the molecular findings confirmed the results of the ampelographic description. More importantly, they indicated that only the combination of the ampelographic description with molecular markers can allow for a more effective identification and discrimination of grapevine cultivars.

Competitive yeast action against Aspergillus carbonarius growth and ochratoxin A production

The aim of the present study was to investigate the interaction between 67 different yeast isolates and 3 wild isolates of Aspergillus carbonarius originated from Greek vineyards and characterized by high ochratoxigenic potential. The selected fungi were used either as single cultures or combined in a mixed culture. Yeasts and fungi were grown as mono-cultures and co-cultures in solid (MEA, CYA) and liquid (CY broth) media, grape berries and sterilized grape juice. Fungal growth was monitored by means of colony area measurements. The model of Baranyi and Roberts was further fitted to growth data to provide estimates of the colony area growth rate (cm2/day). Moreover, OTA analysis was undertaken for CY broth and agar as well as for grape berries and juice, on the 8th and 15th days of incubation at 25 °C. A significant reduction in fungal growth rate, final colony size and toxin production was observed in both liquid and solid media by the different yeast species assayed. The most competitive strains belonged to Saccharomyces, Pichia, Metschnikowia, Dekkera and Rhodotorula genera. Similar results were obtained from inoculated grape berries and grape juice. Specifically, Saccharomyces cerevisiae Y33 resulted in a decrease in fungal colony area of >90% and 93% after 3 and 4 days of co-culture, respectively. Similar results were obtained for OTA, where toxin concentration of the highest producer (A. carbonarius F3) was reduced from 14,983 and 31,565 ng/mL at 8 and 15 days, respectively, to 5 ng/mL and below detection limit (1 ng/g) when co-cultured with S. cerevisiae Y33. The results of this study could provide a pool of yeast species that must be further investigated for potential application as biological control agents at pre- and post-harvest level in wine and grape juice processing.

Polyphenolic compounds and antioxidants of skin and berry grapes of Greek Vitis vinifera cultivars in relation to climate conditions

The Greek vineyard is home to many minor, indigenous grape cultivar whose diverse polyphenolic content has remained largely unexploited. The study aimed at assaying and assessing the polyphenolic content and antioxidant capacity of: (a) five obscure cultivars; (b) Six biotypes of cultivar ‘Liatiko’; (c) Five prominent cultivars; and (d) three French varieties included for comparison reasons. Results revealed all samples exhibiting high polyphenol content and antioxidant capacity levels. ‘Vertzami’ recorded the highest concentrations in berry skins total anthocyanins, flavanols, flavonols, antioxidant capacity. ‘Mandilaria’ scored the highest value in seed total flavanols compared to all biotypes and cultivars. All ‘Liatiko’ biotypes scored the highest concentrations in seed total flavonoids and flavonols. ‘Liatiko’ and its biotypes exhibited different polyphenolic profiles between them. Owing to the climate change, those indigenous varieties’ substantial polyphenol amounts, and the differences between biotypes, will allow viticulturists to select the varieties/biotypes most appropriate for obtaining higher quality products.

First Report of Grapevine Virus E and Grapevine Virus F in Grapevine in Greece

Grapevine virus E (GVE) and Grapevine virus F (GVF) are members of the genus Vitivirus (family Betaflexiviridae) (Al Rwahnih et al. 2012; Nakaune et al. 2008) with a worldwide distribution. Even though grapevine viruses A and B, two other vitiviruses, are prevalent in Greek vineyards, no other member of the genus Vitivirus has been reported in Greece so far. In 2017, during a study of the virome of Greek grapevines, one sample (D2.1) from cultivar Dafnia (Institute of Grapevine, Likovrisi, Attiki) was subjected to high-throughput sequencing of total RNA extracted from phloem scrapings using a Plant/Fungi Total RNA Purification Kit (Norgen Biotek Corporation, Canada) on an Illumina NextSeq platform (Lifesequencing, S.L., Spain). The run yielded ∼51 million 150-bp paired-end reads. De novo assembly of these reads and subsequent BLAST (n/x) analysis of the produced contigs revealed sequences of GVE and GVF, among others. Almost complete genomes from both viruses were reconstructed with 71 to 98% and 86 to 89% nucleotide sequence identities to GVE and GVF isolates for which sequences are deposited in databases, respectively. The reconstructed genomes were deposited in GenBank with accession numbers MK490829 for GVE (isolate D2-1/8) and MK490830 and MK490831 for GVF (two different GVF variants coinfecting D2.1, namely D2-1/9, D2-1/13). To confirm the presence of these two viruses in the D2.1 sample, two sets of primers (GVEup [5′-ATGGAGTCAAAAGCGATCMG-3′] and GVEdo [5′-ACCTGTGACTGAGCATCAAATAC-3′]; and GVF_F_4521 [5′-TGTGTGGGCKAARACATA TG-3′] and GVF_R_ 5190 [5′-ATCAGAAAAGATGCTMCTCACCT-3′]) were used to amplify a 574 and a 670-bp fragments from the coat protein and polymerase gene of GVE and GVF, respectively. Sanger sequencing of the amplicons confirmed the presence of both viruses in the analyzed sample. Seventy additional samples from the Institute’s collection were screened for the presence of GVE and GVF, and the viruses were identified in five and 12 vines, respectively. To our knowledge, this is the first report of GVE and GVF in grapevine in Greece.

Assessment of genetic diversity of Vitis vinifera local cultivars of northern Greece as a means for valorization of vine and wine territories

Grapevine cultivation and wine production occur in Greece since antiquity. Modern wineries have focused on the cultivation of international varieties; however, a marked trend to the revival of the local grapevine varieties has been noted in the last two decades underlying the confidence and the intention to invest on the local plant resources aiming to produce unique products. This brings up the urgent necessity to genetically identify the local varieties that constitute the Greek vineyard so as they will be available for further development. Simple-Sequence Repeats (SSRs) molecular markers, including those proposed by the International Organization of Vine and Wine (OIV) as molecular descriptors, have been used to genotype: i) 12 wine producing varieties maintained in the ex-situ ampelographic collection of the Greek Gene Bank (GGB), ii) 30 varieties that have been collected from various sites of northern Greece in order to verify and confirm their identity, and iii) 11 international varieties that have been used as references. The final dendrogram displays the genetic closeness between the analysed varieties, bringing up cases of synonymies and homonymies.

Study of Some Qualitative and Quantitative Characters of the Grapes of Indigenous Greek Grapevine Varieties (Vitis vinifera L.) using HPLC and Spectrophotometric Analyses

Grape skins and seeds are sources of various quality characters of grapevine varieties, such as phenolic compounds, anthocyanins, resveratrol etc. The Greek vineyard is characterized by a large number of indigenous grapevine varieties which remain almost unexploited in terms of their different phenolic content. The aim of this study was to assay the polyphenolic content of seven red native Greek grapevine varieties (‘Kolliniatiko’, ‘Bekari’, ‘Bakouri’, ‘Kountoura mavri’, ‘Doubrena mavri’, ‘Thrapsa’, and ‘Mavro Spetson’) using high performance liquid chromatography (HPLC) coupled with a diode array detector and spectrophotometer. All studied varieties were cultivated in the same area and under the same conditions. The results revealed that despite the fact that anthocyanins and phenolic compounds content were in high levels for all varieties, statistically significant differences were recorded among them. The highest anthocyanins concentration was recorded in ‘Kolliniatiko’, while the lowest anthocyanins concentration was recorded in ‘Mavro Spetson’. Grapevine variety ‘Mavro Spetson’ presented the highest concentration of total phenolic compounds, with statistically significant difference compared to ‘Kountoura mavri’ and ‘Bekari’. Grapevine varieties ‘Kolliniatiko’ and ‘Bekari’ presented the highest concentrations in trans-resveratrol and piceid content, while grapevine variety ‘Doubrena mavri’ presented the highest concentration in vanillin content. These unexploited rare native cultivated varieties contained appreciable amounts of non-colored phenols as well as anthocyanins, meaning that they would be worthy of further study and use for the production of quality wines.

Sustainable bioeconomy transitions: Targeting value capture by integrating pyrolysis in a winery waste biorefinery

In the transition to a sustainable Bioeconomy, pyrolysis-based bioenergy is finding a new impetus within integrated biorefinery concept, which is one of the vehicles of this transition. An integrated winery waste biorefinery is proposed in this study. A closed-loop approach was designed, by integrating pyrolysis process, to the primary pomace (winery waste) biorefinery. The results of the experimental study revealed that from 15 t of fresh grapes of the special variety ‘Xinomavro’ harvested per hectare in Northern Greek vineyards, along with the 10.5 t of red wine (main product), 0.27 t of hydrocolloids and 0.06 t of grape-seed oil can be produced, via pomace extraction. By pyrolyzing the remaining solid wastes derived from pomace extraction, in the secondary biorefinery, 0.52 t of biochar, 0.80 t of bio-oil and 0.630 MWh energy, can be produced. It was resulted that pyrolysis of the primary biorefinery wastes brings many benefits: it increases the number of the biorefinery products to 5 by shifting from a mono to multi-product pathway, creates an additional economic benefit of 4470€ ha−1 and avoids 355 kg CO2/t on dry pomace basis. The pyrolysis biochar can be used as a soil improver, because it contains N, P, K, Ca nutrients, significant amounts of K, Na, Mg, Ca, P, Fe, Zn, and negligible amounts of heavy metals. With the present study at TRL 4, protocols for the winery waste-biorefinery processes were created and a pathway for sustainable waste management, in the wine industrial sector, with environmental and socio-economic benefits, was provided. Pyrolysis-based bioenergy production integrated in the biorefinery was studied, as a paradigm for the greater integration of bioenergy into the Bioeconomy. Within the wider Bioeconomy transition, it is important to move from sustainability debate into sustainable solutions, and bridge the gap between research and industrial implementation.

Monitoring the Temporal Expression of Genes Involved in Ochratoxin A Production of Aspergillus carbonarius under the Influence of Temperature and Water Activity.

The objective of this study was to investigate the effect of environmental factors, namely temperature and water activity, on genes involved in the regulation of ochratoxin A (OTA) production over time. For this purpose, the previously characterized toxigenic Aspergillus carbonarius Ac29 isolate from Greek vineyards and the A. carbonarius ITEM 5010 reference strain were subjected to combined temperature and water activity (aw) treatments to study OTA production and relative gene expression. The fungal isolates were grown on a synthetic grape juice liquid medium (SGM) under different temperature (20 °C, 25 °C and 30 °C) and aw (0.94 and 0.98) regimes. The expression of the AcOTApks, AcOTAnrps, and laeA OTA related genes was investigated using real time PCR. Gene expression was monitored at the same time points, along with fungal biomass and OTA accumulation at three, six and nine days of incubation. In gene expression analysis, stimulation of the biosynthetic genes was observed a few days before any toxin could be detected. This fact may underline a possible early indicator of potential toxin contamination of grapes. However, the transcript levels varied with respect to the different combinations of ecophysiological conditions and time, highlighting a complex regulation of OTA related gene expression of A. carbonarius in the specific medium.