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Numerous grapevine breeding programs are carried out in the world with the aim of getting varieties resistant to fungal diseases. The basics are often native varieties in order to preserve the authenticity of a certain area. Croatia counts more than a hundred native varieties but there is no research on their downy mildew susceptibility, which is essential for further breeding. The aim of this study is to compare the two most widely used methods for estimating the susceptibility of grapevine varieties: in vivo (field trial) and in vitro using the leaf disk assay. The research was conducted on nine genotypes: seven native varieties, Cabernet Sauvignon and Vitis riparia. In vivo disease symptoms were monitored after flowering four times in two-week intervals, and the leaf disks were evaluated once on the leaves of different developmental stages (from the 1st to 7th leaf from the shoot tip). Significant differences in susceptibility between the examined genotypes were found using both methods. By using the in vivo method significant differences were noticed during the initial terms, while the differences were reduced later on suggesting that the rate of disease development is peculiar to the genotype. In vitro method showed, together with differences between the genotypes, significant differences between the leaves of different developmental stages. The highest level of correlation between the results of these two methods (r>0, 5) was determined between the third term in vivo and the 3rd to the 6th leaf (from the shoot tip) in in vitro conditions.

Context and purpose of the study – Croatia has rich grapevine genetic resources with more than 125 autochthonous varieties preserved. Coastal region of Croatia, Dalmatia, is well known for wine production based on autochthonous grapevine varieties. Nevertheless, only couple of these are widely cultivated and have greater economic importance. Grape seeds are sources of polyphenols which play an important role in organoleptic and nutritional value of grape and wine. Hence, the aim of this study was to evaluate the simple polyphenols from grape seeds in 20 rare autochthonous grapevine varieties. Material and methods – Samples were collected during two consecutive years (2011. and 2012.) from germplasm collection in Split (Dalmatia). Grape samples were constituted of five bunches of fully ripe grapes. Seeds were manually separated, freeze-dried, grounded and stored at a low temperature until analyses. Polyphenolic compounds were analysed using HPLC analysis. Results – Eight polyphenolic compounds, galic acid, monomeric flavan-3-ols (catechin, epicatechin, gallocatechin and epicatechin 3-O-gallate) and procyanidin dimers (B1, B2 and B4) were detected. According to the investigated polyphenolic compounds significant differences between investigated varieties were found. Gallic acid content ranged from 91.0 to 245.08 total monomeric flavan-3-ols from 619.2 to 13539.6 mg kg-1 and total procyanidin dimers from 975.3 to 4140.2 mg kg-1 of seed. Catechin (263.2 to 8124.2 mg kg-1 seed) was found as main monomeric flavan-3-ol, epicatechin 3-O-gallate, gallocatchin and epicatechin varied between 0-164.31, 37.19-155.07 and 277.5-5224.4 mg kg-1 seed, respectively. Procyanidin B2 (420.2 to 2207.8 mg kg-1 seed) was found as a main procyanidin dimer. Procyanidin B1 and B4 amount varied between 401.80-165.19 and 276, 7-1539.4 mg kg-1 seed, respectively. Gegić had lowest and varieties Plavac mali and Babić highest amount of all investigated polyphenolic compounds. This study presents the first evaluation of Croatian grapevine varieties by characterization of seed polyphenolic compounds and it shows huge variability among them. More detailed analysis of polyphenolic compounds in selected varieties are carry out in our further research activities.

INTRODUCTION – Cryopreservation is a method for long-term conservation in which explants are stored at the ultra-low temperature of liquid nitrogen. In the case of grapevine, cryopreservation protocols have not been defined enough for wide application to numerous cultivars. Cryotherapy is a novel technique, which eliminates plant pathogens during exposure to ultra low temperatures. Plant cryopreservation is a complex procedure during which explants are exposed to potentially stressing events. Genetic stability of regenenerated plants is a part of cryopreservation protocol establishment. AIMS AND SCOPES – The main goals of this research are: to determine the efficient cryopreservation protocol for Croatian native grapevine cultivars and international cultivars, to test efficiency of cryotherapy in virus eradication and to check genetic stability of regenerated plants. This research was performed aiming to introduce this technique in general viticulture. MATERIALS AND METHODS – In vitro explants of Croatian (Plavac mali, Maraština-(plus their infected genotypes), Pošip and Škrlet) and international cultivars (Chardonnay, Cabernet Sauvignon, Merlot and Pinot Noir, within their infected genotypes) were taken for cryopreservation experiment, which had been established previously on cultivar Portan. Droplet- vitrification protocol was performed. Regenerated two-months in vitro plants from infected genotypes (control and cryopreserved apices) were subjected to ELISA testing. AFLP markers were employed to test genetic stability on cultivar Portan. Eight AFLP primer combinations were used on cryopreserved and non-cryopreserved shoot tips (sampled after sucrose preculture, loading and treatment with half-strength PVS2 solution) for a total of 43 plants tested. RESULTS AND DISCUSSIONS –The basal cryopreservation protocol, performed on international cultivars revealed: intermediate regrowth was achieved with Chardonnay (30%) and high with Merlot (70%), before and after cryopreservation, which was comparable to previously published results on 10 Vitis cultivars, which produced an average recovery of 64%. Similar results were obtained with healthy and virus infected cultivars. The same protocol, performed on Croatian cultivars, showed a low survival and recovery with only one (Maraština) of the four cultivars tested. This low success might be due to the fact that cryopreservation experiments were performed using cultures which had been introduced in vitro very recently and that the in vitro culture conditions employed were not adapted to the multiplication of these Croatian cultivars. The results of the cryotherapy experiments revealed that a high proportion of Chardonnay (100%) and Cabernet Sauvignon (78%) plants were virus-free after being subjected to cryopreservation. Control plants showed a high proportion of healthy plants, almost the same as with LN exposed ones for both viruses. AFLP markers revealed no significant differences in AFLP profiles after sucrose preculture, treatment with the loading solution and with half-strength PVS2, whereas an increase in polymorphic fragments was observed in non- cryopreserved and cryopreserved samples treated with PVS2 solution, the number of polymorphic fragments increasing with longer durations of exposure to PVS2 solution. CONCLUSIONS AND POSSIBLE APPLICATIONS – Cryotherapy as a method can be efficient for virus eradication even in the preconditioning steps of a cryopreservation protocol. A genetic stability study should be included in post- cryopreservation experiments.

INTRODUCTION – Before Philloxera crisis in Croatia were more than 400 grape varieties under cultivation, but today their number is drastically decreased. Recently, many efforts in the preservation of grapevine biodiversity have been undertaken: detailed inventory of wine-growing regions, establishing of National and regional collections and their systematic ampelographic and genetic characterisation. Recent genetic research resulted in revealing synonyms and homonyms, as well as genetic relationship within Croatian native cultivars, as well as with cultivars from other regions. Evaluation of quality traits of native cultivars has also started. AIMS AND SCOPES – The main goals of this research are: to determine the number of Croatian native grapevine cultivars based on inventarisation of viticultural areas, ampelographic and genetic characterization ; to perform detailed ampelographic characterization using OIV descriptors ; to perform the genetic characterization of Croatian native grapevine cultivars using SSR markers and to determine the level of genetic variability within Croatian native grapevine cultivars. MATERIALS AND METHODS – Vines of a potentially native cultivars ware selected and marked and used as a source of propagating material for establishment of a National collection of Croatian grapevine cultivars located at experimental station Jazbina, at Faculty of Agriculture, University of Zagreb. In this process more than 140 accessions were introduced to this collection. Phenotyping of collected accessions was performed using 47 OIV descriptors for grapevine cultivars and Vitis species that were selected within the GrapeGen 06 and used for cultivars description in European Vitis database. SSR genotyping was performed using a total of nine markers. Six SSR markers (VVS2, VVMD5, VVMD7, VVMD27, VrZAG62, VrZAG79) recommended by OIV and three additional ones (VVMD25, VVMD28, VVMD32) selected during the Grapegen06 project. RESULTS AND DISCUSSIONS – Based on results of inventarisation, ampelographic characterization and genetic characterization the list of 103 Croatian native grapevine cultivars is defined together with their synonyms and population status/vulnerability evaluation. Based on presented information on population status of Croatian native grapevine cultivars we can conclude that more than 50% of cultivars are critically endangered, endangered or vulnerable. The results of ampelographic characterization of Croatian native cultivars are showing the high level of variability. Comparison of Croatian cultivars SSR profiles with the results of the other cultivars in EU- vitis database confirmed that majority of them are unique and exist only in Croatia. Since most of them are duplicated in grapevine germplasm collections throughout Croatia they are considered as “saved”. Ongoing genetic studies with increased number of SSR loci (>30) as well as use of SNP markers will allow us more detail analysis of genetic structure and relatedness of Croatian native cultivars. CONCLUSIONS AND POSSIBLE APPLICATIONS – The preserved native cultivars are presenting a good base for future development of Croatian viticulture and because of high genetic variability, as well as some traits that are detected within Croatian native cultivars they could be of interest to other countries for production as well as for breeding.

There is a large number of grapevine cultivars grown in Croatia. Most of them are considered to be autochthonous. Unfortunately, clonal selection of these cultivars has never been done and there is no planting material of adequate quality standards. As a first step in clonal selection, mass positive selection together with sanitary selection of 15 economically important cultivars has been started. In rpoceeding of genetic selection visual symptoms of the viruses have also been observed. Samples of dormant canes have been taken from vines with positive agronomic traits and no visible symptoms of virus infection. The presence of four economically important viruses was evaluated using ELISA (enzyme linked immunosorbent assay): two nepoviruses, Grapevine fanleaf virus (GFLV) and Arabis mosaic virus (ArMV) and two closteroviruses Grapevine leafroll-associated virus 1 (GLRaV-1) and Grapevine leafroll-associated virus 3 (GLRaV-3). Survey of virus deseases in production vineyards of different vine-growing region of Croatia has disclosed the widespread presence of viruses and the number of non infected vines was rather low. This is true especially for the coastal region where it was not possible to find any healthy plant of certain cultivars. Although no visible symptoms were observed , it was not rear to detect multiple infections with two or even three viruses. Mostly spread virus in Dalmatia was GFLaV-3 with approximately 80% of vines infected. The situation is much better in northern region, where almost half analysed plants were healthy and most represented virus is GLRaV-1. The presence of both nepoviruses is rather low, especially ArMV (only 1% of vines infected) and somewhat higher of GFLV in Dalmatia.

At the end of 19th and beginning of 20th century in Croatia were, according some literature data, more than 400 grape varieties. Unfortunately, because of many reasons (introduced pests and pathogens, modern economic pressure and introduction of world well known cultivars), drastic erosion of autochthonous cultivars has occurred. In last decade, many efforts in the preservation of grapevine biodiversity in Croatia have undertaken. Detail inventarisation of vine-growing regions in Croatia has shown that lot of autochthonous cultivars still can be found, but many of them are endangered and for a long time neglected. To preserve them against the extinguishing, we have established since 2001 national ex situ collection at the Faculty of Agriculture in Zagreb, where we have collected about 100 autochthonous accessions so far. Besides, we started to build few regional collections, to save duplicates and enable their economic evaluation in appropriate climatic conditions. Besides multiplication and planting into collection, amphelographic description (OIV descriptors) and genetic analyses (at least 6 core SSR loci) of cultivars have been carrying out. So far, about 60 cultivars have known genetic profile, and all others are in the progress. Comparison of the microsatellite genotypes of the analyzed cultivars with international varieties shows that most of them have unique genotype which supports hypothesis they are Croatian autochthonous cultivars. In few cases microsatelites were also useful in clarification of synonym and homonym questions as well as parent/progeny relationships. All this data, along with photos from different plant parts, are supposed to be entered in the electronic database. We have observed a high quality potential in case of some neglected cultivars, and we started economic revitalization for few most promising (Malvasija dubrovačka, Crljenak kaštelanski). Beside that, we started with clonal and sanitary selection in case of most important native varieties (Plavac mali, Pošip, Babić, Žlahtina, Kraljevina, Škrlet, Plavina, Debit, Maraština, Vugava).

Among 48 cultivars holding the „ Malvasia“ name listed in the Vitis International Variety Catalogue (http://www.genres.de/idb/vitis/vitis.htm) there are also two cultivated in Croatia – „ Malvazija istarska“ and „ Malvasia dubrovačka“ . Since recently it is known that Malvasia dubrovacka is not a unique local cultivar (genotype) but the member of the synonym group which is scattered across the Mediterranean and even at the Atlantic islands (Malvasia delle Lipari, Malvasia di Sardegna, Malvasia de Sitges, Malvasia de Tenerife, ...) (Crespan et al., 2006). So far, Malvazija istarska seems to be a unique genotype spread only in the area of peninsula Istria and north-east of Italy. According to Boursiquot et al. (2007), the real number of Malvasia cultivars is not so large as it used to be believed, i.e. they found that several so called Malvasia cultivars are synonyms of already well known cultivars. Taking into account the historical fact that „ Malvasia wine“ used to be exported from Greece to Venice for centuries and an assumption that the name „ Malvasia“ has Greek origin (according to medieval city Monemvasia), as well as the fact that by vegetative propagation cultivars can be easily disseminated and preserved over the space and time we have hypothesized that Croatian Malvasias might still be grown (under different names) in Greece or some other places along the historical naval routs. We have used an opportunity to participate in the sailing expedition „ Malvasia Mediterranea 2005“ (http://www.malvasia-mediterranea.com/) to explore and sample the tissue for DNA of the cultivars along the eastern coast of Adriatic and Ionian Sea, and south of Peloponnesus. Twenty-eight grapevine samples from Greece (islands and hinterland), more than 30 from Croatia, and several from Italian coastal regions that used to be on the ancient naval routs from Monemvasia to Venice, have been assessed by genomic DNA SSR markers and analyzed in term of genetic similarity. Similarly as in several previous studies we have found a weak relationship of actual Greek cultivars with Croatian and Italian ones. We have found only one original genotype that is nowadays present in Greece, Italy and Croatia under three different names (synonyms), Pavlos, Malvasia lunga (M. del Chianti) and Maraština, respectively.

Borgonja crna is grapevine cultivar (variety) grown in Croatia only on peninsula Istria (north Croatian coast) and has regional importance. Borgonja crna has superior quality in comparison to the other black grapevine cultivars grown in that region. All the available literature data suggests that Borgonja and Gamay noir (Gamay Beaujoilais) could be synonyms for the same variety. This assumption is based on the similarity of phenotype. For that reason even producers started to plant Gamay noir due to the lack of planting material of Borgonja crna. The aim of this work was to examine whether the Borgonja crna and Gamay noir are identical or two different cultivars. In the year 2004 various ampelographic and genetic analyzes were conducted. As a part of ampelographic analysis selected OIV descriptors, uvometry and philometry were applied. Jung leaves from the selected vines were collected, liophilised and DNA extraction was performed according to Doyle and Doyle (Focus, 1990.). Microsatellite markers (SSR) were chosen for plant genotyping. According to the project GENRES 081, 6 core SSR loci and three additional were used. The results shown that, although morphological similar, Borgonja crna and Gamay Beaujolais have different genetic profiles and cannot be considered as synonyms.

Vitis species are shown to be susceptible, tolerant, or resistant to the Plasmopara viticola (Berk. & M. A. Curtis; Berl. & de Toni) pathogen. The obligate, biotrophic, and polycyclic P. viticola oomycete can infect any green organ of the host plant, including the shoots, leaves, inflorescences, tendrils, petioles, and green berries. It can therefore have a devastating effect on the host plant. Moreover, significant economic losses occur when effective control methods are not used when growing susceptible species and varieties. The most widespread and commercially significant Vitis species in the world is the grapevine (Vitis vinifera L.). Although the majority of grapevine varieties are susceptible to downy mildew, there are some variations in susceptibility among them. Croatia has a centuries-old heritage of grapevine cultivation. One hundred and twenty-five native varieties were developed and are still grown in Croatia's various geographical and climatic regions. It is therefore considered that in addition to variations in a wide range of biological and economic properties, they also differ in disease susceptibility. Aiming to determine the differences in susceptibility of Croatian native varieties to downy mildew, phenotyping of selected varieties was carried out using the following methods: leaf disc bioassay, measurement of chlorophyll fluorescence and multispectral imaging, and analysis of polyphenolic and volatile organic compounds in grapevine leaves. Polyphenolic and volatile organic compounds were analysed in leaves before and 24, 48, and 96 hours after inoculation with P. viticola suspension. Measurement of chlorophyll fluorescence and multispectral imaging was performed before inoculation and during six terms after inoculation. The leaf disc bioassay proved to be effective for classifying grapevine varieties into the OIV resistance classes to downy mildew. In addition, parameters of chlorophyll fluorescence, such as Fv/Fm, Fq'/Fm' and ETR, were useful in distinguishing infected from non-infected leaf discs shortly after inoculation, that is, before the appearance of visible symptoms of the disease. Moreover, the Fq'/Fm' and qP parameters helped distinguish between varieties with different susceptibility to P. viticola. The results of the polyphenolic compound analysis clearly distinguished the native varieties into the OIV resistance classes. Resistance was found to be dependent on the composition and content of polyphenolic compounds present in the leaves before inoculation. In all terms following the P. viticola inoculation, the stilbene resveratrol-3-O-glucoside content was higher in infected leaves compared to uninfected. Finally, volatile organic compound analysis can be used to distinguish infected from noninfected samples as well as resistant control genotypes (Solaris, Vitis riparia Michx.) from V. vinifera varieties. Nevertheless, using this approach does not allow for the division of native varieties into the OIV resistance classes. Less susceptible native grapevine varieties that belong to OIV class 5 (Malvazija istarska, Ranfol, and Teran) could be interesting to use in breeding programs aiming to produce highquality genotypes tolerant to main fungal diseases. Vrste roda Vitis (loze) osjetljive su, tolerantne ili otporne na uzročnika plamenjače Plasmopara viticola (Berk. & M. A. Curtis; Berl. & de Toni). Vinova loza (Vitis vinifera L.) najuzgajanija je vrsta loza u svijetu iako je većina sorata vinove loze osjetljiva na plamenjaču. Ova bolest ima izrazito devastirajuće djelovanje kad se prilikom uzgoja ne primjenjuju kemijska sredstva za zaštitu bilja. Oomiceta P. viticola je obligatni, biotrofni i policiklički patogen koji može inficirati sve zelene organe biljke domaćina, kao što su mladice, listovi, cvatovi, vitice, peteljke i zelene bobe. Optimalni uvjeti za rast i razvoj ovog patogena su visoka vlažnost i umjerena temperatura zraka. U takvim uvjetima patogen razvija nekoliko ciklusa vegetativnog razmnožavanja, uzrokujući značajan pad prinosa i kvalitete grožđa. Zbog toga je primjena fungicida neizbježan tehnološki postupak pri uzgoju osjetljivih sorata vinove loze, iako te agrokemikalije mogu štetno djelovati na okoliš. Plasmopara viticola podrijetlom je s američkog kontinenta pa su američke vrste (Vitis labrusca L., Vitis riparia Michx., Vitis rupestris Scheele i Muscadinia rotundifolia Small) koevolucijom na istom području razvile visoku ili potpunu otpornost. Donedavno se smatralo da je europska loza V. vinifera neotporna na plamenjaču i pepelnicu, odnosno da ne postoji značajna genetska varijabilnost u otpornosti unutar sorata vinove loze. Međutim, u novije su vrijeme kod nekih od njih, kao što su 'Kishmish vatkana', 'Dzhandzhal kara' i 'Mgaloblishvili', identificirani geni otpornosti, što ih čini vrijednim izvorom gena za oplemenjivačke programe s ciljem stvaranja visokokvalitetnih sorata otpornih na ekonomski najznačajnije bolesti. Pretpostavlja se da, zbog višestoljetnog uzgoja vinove loze u Hrvatskoj i njezine prilagodbe specifičnim okolišnim uvjetima, među hrvatskim autohtonim sortama također postoje razlike u osjetljivosti na bolesti. S ciljem utvrđivanja osjetljivosti hrvatskih autohtonih sorata na plamenjaču, provedeno je istraživanje koje je obuhvatilo odabrane sorte te nekoliko otpornih i osjetljivih kontrolnih genotipova koristeći sljedeće metode fenotipizacije: metoda lisnih diskova, mjerenje fluorescencije klorofila i multispektralno snimanje lisnih diskova te analiza polifenolnih i hlapljivih organskih spojeva u listovima. Metoda lisnih diskova provedena je u kontroliranim laboratorijskim uvjetima sukladno deskriptoru 452-1 Međunarodne organizacije za vinovu lozu i vino (engl. International Organisation of Vine and Wine, OIV). Na temelju toga, svaki je genotip svrstan u odgovarajući razred otpornosti pri čemu su genotipovi svrstani u razred 1 najosjetljiviji, a oni svrstani u razred 9 potpuno otporni na plamenjaču. Mjerenje fluorescencije klorofila i multispektralno snimanje provedeno je pomoću instrumenta CropReporterTM tijekom sedam termina. Prvo mjerenje provedeno je na netretiranim lisnim diskovima, a završno kad su se razvili vidljivi simptomi bolesti (sporulacija). Polifenolni spojevi analizirani su tekućinskom kromatografijom visoke djelotvornosti (engl. high-performance liquid chromatography, HPLC), a hlapljivi organski spojevi primjenom vezanog sustava plinski kromatograf-spektrometar masa (engl. gas chromatography-mass spectrometry, GC-MS). U tu svrhu, analizirani su listovi uzorkovani prije tretiranja te 24, 48 i 96 sati nakon inokulacije P. viticola suspenzijom. Lisni diskovi i listovi inokulirani su suspenzijom koncentracije 2 × 105 spora/mL raspršivanjem po naličju. Suspenzija je dobivena umakanjem listova sa svježe razvijenom sporulacijom u ultračistu vodu do zamućenja. Utvrđeno je da je metoda lisnih diskova učinkovita za razvrstavanje sorata vinove loze u OIV razrede otpornosti na uzročnika plamenjače vinove loze. Parametri fluorescencije klorofila, odnosno Fv/Fm, Fq'/Fm' i ETR, razlikovali su inficirane od neinficiranih lisnih diskova, a pomoću parametara Fq'/Fm' i qP moguće je razlikovati slabije i jače osjetljive sorte ubrzo nakon zaraze, odnosno prije pojave vidljivih simptoma bolesti. Rezultati analize polifenolnih spojeva jasno su razdvojili autohtone sorte u razrede otpornosti pri čemu je utvrđeno da otpornost ovisi o sastavu i sadržaju polifenolnih spojeva u listovima prije inokulacije. Povećan sadržaj stilbena resveratrol-3-O-glukozida razlikovao je inficirane od neinficiranih listova tijekom svih termina nakon inokulacije. Sadržaj piceatanola i ukupnih stilbena razlikovao je potpuno otporan OIV razred 9 (Vitis riparia Michx.) od ostalih OIV razreda čime su potvrđena njihova snažna antimikrobna svojstva. Na temelju polifenolnih profila V. vinifera sorata, utvrđeno je da su uglavnom flavonol glikozidi odgovorni za manju osjetljivost sorata koje sadrže višu koncentraciju ovih spojeva. Analizom hlapljivih organskih spojeva moguće je razlikovati otporne kontrolne genotipove od V. vinifera sorata kao i inficirane od neinficiranih uzoraka. Iako ovom metodom nije moguće razdvojiti autohtone sorte u razrede otpornosti, rastući sadržaj nekoliko detektiranih hlapljivih spojeva nakon inokulacije zajedničko je svojstvo manje osjetljivih autohtonih sorata i otpornih kontrolnih genotipova ('Solaris', Vitis riparia Michx.). Manje osjetljive autohtone sorte koje pripadaju OIV razredu 5 ('Malvazija istarska’, ‘Ranfol’ i ‘Teran’) mogle bi biti zanimljive za korištenje u oplemenjivačkim programima kojima je cilj proizvesti visokokvalitetne sorte tolerantne na najznačajnije gljivične bolesti.