Silvio Šimon

Chronic fungal meningitis caused by Aureobasidium proteae

We present a case of chronic meningitis due to the mold Aureobasidium proteae. Clinical features, the disease course, as well as the diagnostic methods and optimal treatment options are discussed. This case confirms the neuroinvasiveness of A. proteae and introduces it as a new human pathogen.

Plum germplasm in Croatia and neighboring countries assessed by microsatellites and DUS descriptors

At a certain period during the last century, former Yugoslavia (which among others used to include Bosnia and Herzegovina, Croatia, and Serbia) was the biggest producer of plums in the world. Traditional plum cultivars, still grown in this region, represent a mixture of several species including: European plums (Prunus domestica L.), mirabelles (Prunus insititia var. syriaca (Borkh.) Koehne), and damsons (P. insititia L.). The basic problem with the utilization of this plum germplasm, either for cultivation or breeding purposes, is a lack of reliable pomology data or reference repositories that would enable positive identification of cultivars. In this study, 62 plum accessions (42 traditional Croatian accessions, six well-known traditional accessions collected from Serbia and Bosnia, and 14 international, reference cultivars) were assessed using microsatellite markers and distinctness, uniformity, and stability (DUS) plum descriptors. Nine primer pairs amplified 168 distinct alleles, or on average 18.7 alleles per locus. A significant differentiation between the traditional plum cultivars and international reference cultivars, was detected through Fst (Fst = 0.022; P < 0.0001), analyses of molecular variance (AMOVA; fCT = 0.054; P < 0.05) and later confirmed by a factorial correspondence analysis (FCA). Bayesian method enabled the classification of mirabelle, damson, and European plum genotypes. Principal component analyses, based on 22 morphologic traits, managed to separate mirabelle accession from the European plum and damson accessions, but there was a general lack of correlation between the observed morphologic traits and the molecular data. Results of this study indicate that traditional Croatian accessions represent a diverse and underutilized plant genetic material, which should be conserved.

Whole genome amplification and microsatellite genotyping of herbarium DNA revealed the identity of an ancient grapevine cultivar

Reconstruction of the grapevine cultivation history has advanced tremendously during the last decade. Identification of grapevine cultivars by using microsatellite DNA markers has mostly become a routine. The parentage of several renowned grapevine cultivars, like Cabernet Sauvignon and Chardonnay, has been elucidated. However, the assembly of a complete grapevine genealogy is not yet possible because missing links might no longer be in cultivation or are even extinct. This problem could be overcome by analyzing ancient DNA from grapevine herbarium specimens and other historical remnants of once cultivated varieties. Here, we present the first successful genotyping of a grapevine herbarium specimen and the identification of the corresponding grapevine cultivar. Using a set of nine grapevine microsatellite markers, in combination with a whole genome amplification procedure, we found the 90-year-old Tribidrag herbarium specimen to display the same microsatellite profile as the popular American cultivar Zinfandel. This work, together with information from several historical documents, provides a new clue of Zinfandel cultivation in Croatia as early as the beginning of fifteenth century, under the native name Tribidrag. Moreover, it emphasizes substantial information potential of existing grapevine and other herbarium collections worldwide.

Genetic Diversity of Wild Grapevine [Vitis vinifera L. subsp. sylvestris (Gmel.) Hegi] in the Eastern Adriatic Region

The genetic diversity of wild grapevine [Vitis vinifera L. subsp. sylvestris (Gmel.) Hegi] in the eastern Adriatic region, which includes Croatia and Bosnia and Herzegovina, has previously not been documented. Natural populations of wild grapevine were identified in their natural habitats, and their genetic diversity was studied and compared with both local and widespread European cultivars. Ninety-two grapevine genotypes were determined at 21 nuclear microsatellite loci, including 53 wild grapevine individuals, 32 diverse local Croatian and west European cultivars, and seven commonly used rootstocks. Two hundred thirty-nine alleles were detected, with a mean of 11.4 alleles per locus and high heterozygosity, ranging from 0.461 to 0.897 for observed heterozygosity and from 0.391 to 0.837 for expected heterozygosity. The genetic diversity of the wild grapevines was slightly lower than that observed in cultivars. Distance- and model-based cluster analysis differentiated three main groups, indicating clear separation between wild, cultivated, and rootstock accessions. This study greatly contributes to knowledge of genetic diversity in local wild grapevine populations in Croatia and Bosnia and Herzegovina, and provides necessary information for their conservation and further characterization.

Intravarietal agronomic variability in croatian native "Vitis vinifera" L. Cultivar Grk with female flower and seedless berries

The Croatian native, Vitis vinifera L. cv. Grk, is one of only a few wine cultivars with a female flower, resulting in two types of berries in the cluster: fully developed seeded (~3.5 g) and undersized seedless (~0.7 g), both ripe at harvest. This study investigated the intravarietal variability of Grk and the impact of the different proportion of seedless berries on grape yield and quality of Grk clones. Significant differences in cluster proportions of seedless berries among Grk clone candidates were determined. A high positive correlation was found between the seedless berry proportion and single berry weight, must tartaric acid content, and free volatile terpenes. These results indicate that it is possible to select clones with a higher proportion of seedless berries, which can then be used to improve the wine quality of Grk.

Distribution and partial molecular characterization of Grapevine leafroll-associated virus 2 (GLRaV-2) found in Croatian autochthonous grapevine (Vitis vinifera L.) germplasm

Study of Grapevine leafroll-associated virus 2 (GLRaV-2) distribution was conducted on 13 Croatian autochthonous grapevine cultivars or cultivars which are supposed to be autochthonous included in clonal selection: Babica, Babi, Glavinuša, Grk, Ljutun, Maraština, Mladinka, Ninčuša, Plavina, Plavac mali, Pošip, Vlaška and Vugava. All of them are grown in the southern part of Croatian coastal region (Dalmatia). Sampling was done during autumn 2007, and only a few samples of cultivar Babi were also collected during 2008. All samples were tested for the presence of GLRaV-2 using DAS-ELISA test. Out of 1100 tested grapevine accessions, the presence of GLRaV-2 was determined in 45 samples (4.1%). No virus was detected in seven grapevine varieties (Grk, Ljutun, Mladenka, Ninčuša, Plavina, Pošip, Vlaška). Minimal infection rate was determined in the cultivar Vugava (0.8%) while the most infected cultivar was Babić in which out of 98 analyzed grapevine accessions 33 (33.7%) were positive. All virus-positive tested vines contained mixed infections of several viruses and no vine was infected by GLRaV-2 only. In 14 different, randomly selected grapevine accessions the presence of GLRaV-2 was also confirmed by RT-PCR using the GLR2CP1/GLR2CP2 primer pair. RT-PCR products from four different grapevine accessions were sequenced. Sequences of gene segment coding for the viral coat protein revealed between 97.9% and 99.6% similarity on the nucleotide level, with absolute similarity on amino-acid level. Sixteen grapevine accessions were selected for transmission to herbaceous host Nicotiana benthamiana, but GLRaV-2 was not successfully transmitted to this host. To our knowledge this is the first report of GLRaV-2 in Croatia.ZusammenfassungDie Studie über die Verbreitung von rapevine leafroll-associated virus 2 (GLRaV-2), wurde an den folgenden 13 kroatischen autochtonen oder als autochton geltenden Rebsorten durchgeführt: Babica, Babić, Glavinusa, Grk, Ljutun, Maraština, Mladinka, Ninčuša, Plavina, Plavac mali, Pošip, Vlaška und Vugava. Alle diese Rebsorten werden im südlichen Teil der kroatischen Küstenregion (Dalmatien) angebaut und es werden derzeit Klone von ihnen entwickelt. Die Probennahme wurde im Herbst 2007 vorgenommen, nur einige Proben von der Rebsorte Babi wurden erst im Laufe des Jahres 2008 genommen. Alle Proben wurden mittels des DAS-ELISA-Tests auf Anwesenheit von GLRaV-2 geprüft. Von den 1100 geprüften Weinreben wurde GLRaV-2 bei 45 Proben (4,1%) festgestellt. Bei sieben Rebsorten wurde dieses Virus nicht gefunden (Grk, Ljutun, Mladenka, Nin uša, Plavina, Pošip, Vlaška). Die niedrigste Infektionsrate wurde bei der Sorte Vugava (0,8%) festgestellt, während bei der meistinfizierten Sorte Babi von den 98 analysierten Proben 33 (33,7%) positiv waren. Alle positiv auf Viren getesteten Rebsorten waren mit verschiedenen Viren mischinfiziert; keine Rebsorte war ausschließlich mit GLRaV-2 infiziert. Bei den 14 verschiedenen, stichprobenweise selektierten Weinrebeproben wurde die Anwesenheit von GLRaV-2 im RT-PCR-Verfahren mittels GLR2CP1/GLR2CP2-Primer-Paaren bestätigt. RT-PCR-Produkte von vier verschiedenen Wein- Akzessionen wurden sequenziert. Für das virale Hüllprotein kodierende Sequenzen waren auf der Nucleotidebene zu 97,7–99,6% und auf der Aminosäureebene zu 100% identisch. Sechzehn Weinrebeproben wurden für die übertragung auf den Pflanzenwirt Nicotiana benthamiana selektiert, jedoch konnte GLRaV-2 nicht erfolgreich auf den genannten Wirt übertragen werden. Unseres Wissens ist dies der erste Nachweis

QTL ANALYSIS OF PATHOGEN RESISTANCE FACTORS AND RIPENING TIME TRAITS IN A GENETIC MAP FROM GRAPEVINE BREEDING LINE GF.GA-47-42 CROSSED TO 'VILLARD BLANC'

Marker-assisted selection is an important tool of current grapevine breeding which aims to improve cultivars for more sustainable viticulture. Markers genetically linked to traits of interest are hence required. These can be identified by QTL (quantitative trait loci) analysis scanning genetic maps elaborated from controlled crosses for marker genotypes correlating with segregating phenotypes. Major traits of interest are resistances to pathogens like powdery and downy mildew (Erysiphe necator resp. Plasmopara viticola) of leaves and fruits. In addition, phenological traits have recently acquired special attention. Breeders wish to select grapes with a better defined ?window? of ripening time as ripening behavior affects both pathogen susceptibility and fruit quality. Ripening time needs to be considered within the context of the climate change debate. To identify genomic regions carrying loci affecting resistance traits and ripening time we constructed a genetic map using 151 individuals from the cross of grapevine breeding line Gf.GA-47-42 (?Bacchus? × ?Seyval?) with ?Villard blanc? (?Seibel 6468? × ?Subereux?). Both parental lines are resistant to powdery and downy mildew and vary considerably in ?veraison? time as an indicator of ripening. While Gf.GA-47-42 is rather early, ?Villard blanc? exhibits middle to late ?veraison? and this trait segregates clearly in the cross. Linkage/recombination analysis for map construction was based on more than 350 simple sequence repeat (SSR)-derived markers. Many of these loci were newly extracted from the Vitis reference genome sequence of PN40024 (http://www.genoscope.cns.fr/externe/GenomeBrowser/Vitis/). In addition, the mapping included 210 newly developed SNP (single nucleotide polymorphisms) markers. A subset of these was analyzed with two different techniques permitting to assess the reliability of SNP genotyping with both methods. The resulting map was scanned for QTL influencing powdery and downy mildew disease resistance and the time of ?veraison?. Several major QTL were identified.

Estimate of intravarietal genetic variation as a prerequisite for successful clonal selection in grapevine

ESTIMATE OF INTRAVARIETAL GENETIC VARIATION AS A PREREQUISITE FOR SUCCESSFUL CLONAL SELECTION IN GRAPEVINE

First Report of Beet yellows virus (BYV) on Sugar Beet in Croatia

Sugar beet (Beta vulgaris var. saccharifera L.) is the main crop used for the production of sugar in Croatia. Beet yellows virus (BYV), type member of the genus Closterovirus, is characterized by flexuous, filamentous particles which are transmitted by several aphid species in a semipersistent manner (Agranovsky and Lesemann 2000). This widely spread virus is reported from most of the sugar beet-growing areas of the world, including Europe. In the geographic region that was once the former Yugoslavia, the presence of BYV was suspected based on field symptoms reported from Serbia (Nikolic 1951) and Croatia (Panjan 1951). During the 2013 and 2014 growing season, the presence of sugar beet plants with symptoms of yellowing was sporadically observed in commercial fields located in eastern Croatia (i.e., Tovarnik, Bosnjaci, and Virovitica). In late July 2014, a significant number of sugar beet plants, located mainly on the field edges, with the aforementioned symptoms were noticed in experimental fields of various sugar beet cultivars located at the University of Zagreb, Faculty of Agriculture. In total, 102 plants collected from commercial fields and 12 plants from the university experimental fields with cvs. Torda and Libero were selected and screened using ELISA for the presence of Beet necrotic yellow vein virus (BNYVV), Beet mosaic virus (BtMV), Beet mild yellowing virus (BMYV), Beet western yellows virus (BWYV), and BYV. To conduct the diagnostic assays, commercial ELISA kits from Loewe Biochemica GmbH (BNYVV, BtMV, and BYV) and Sediag (BMYV and BWYV) were used. BYV was confirmed in five plants from commercial fields and in all the plants from Zagreb. Due to the presence of single infections in plants from Zagreb, and in order to determine the detrimental effects of BYV, all plants from the study field control plots (104 of Torda and 103 of Libero) were tested by ELISA. Tests revealed the presence of BYV in 18 (17.3%) plants of Torda and in 34 (33%) plants of Libero. BYV infections averaged a 9 to 10% reduction of pure root yield and sugar content in both cultivars compared with BYV-free plants. One ELISA-positive plant per cultivar was chosen for the extraction of total RNA using the QIAGEN RNeasy plant mini kit according to manufacturer’s instructions. In both plants, the presence of BYV was confirmed by RT-PCR using four set of diagnostic primers (Kundu and Rysanek 2004). PCR products from both samples, named BYV- CRO-T from Torda and BYV-CRO-L from Libero, were purified and sequenced in both directions (Genbank Accession Nos. KP704263 to KP704268). Nucleotide sequence alignment of the DNA fragments for both Croatian isolates, including the C-terminal part of L-Pro and N-terminal part of MET and HSP70, showed 99.2, 99.7 and 100% similarity, respectively. Nucleotide comparisons of Croatian isolates with Californian (Genbank Accession No. AF056575) showed a 97% similarity for L-Pro and HSP70 and a 99% for MET. To our knowledge, this is the first report of Beet yellows virus in Croatia and its detrimental effect on actual sugar beet cultivars grown under Croatian environmental conditions.

Grapevine variety determination from herbarium and archeological specimens

Genotyping old grapevine samples, in particular cultivar identification via microsatellite profiling, is not yet routine. Success depends on several factors, the age of the investigated material being the most obvious. In addition, the amount and integrity of DNA depends on specimen storage/preservation history. Moreover, conta-mination and fragmentation processes make the isolated DNA a difficult template for PCR amplification. Three old grapevine specimens were investigated in this study. Two derived from a 90-year-old Croatian grapevine herbarium collection: cv. ?Tribidrag? and cv. ?Brzamin?. Both are indigenous Croatian grapevine cultivars not present in contemporary germplasm collections. The third was a 2000-year-old archeological underwater grapevine specimen. Several different approaches for DNA isolation were tested. A commercial plant DNA isolation kit was the best choice in terms of avoiding contamination with contemporary grapevine DNA. Initial attempts to PCR-amplify standard grapevine microsatellite loci from isolated DNA did not work satisfactory. Therefore, a whole genome amplification (WGA) kit was applied to overcome this problem. The WGA performed well with the cv. ?Tribidrag? herbarium sample and the 2000-year-old archeological specimen. While the amplified, cloned and sequenced DNA from the archeological specimen was from origins other than grapevine, indicating contamination of the specimen, clones deriving from the cv. ?Tribidrag? corresponded to different grapevine chromosomes. Therefore, we used the cv. ?Tribidrag? DNA for successful amplification of the VVS2 microsatellite locus by PCR, thereby demonstrating the proof of principle. Eventually we genotyped the cv. ?Tribidrag? specimen at six standard microsatellite loci. The SSR profile obtained was identical to that of ?Zinfandel?/?Primitivo?/?Crljenak kastelanski?. These results are in line with the previously proposed Croatian origin of ?Zinfandel?. This time we identi¬fied the name ?Tribidrag? as the most ancient name for cv. ?Zinfandel?, which has been cultivated in Croatia, according to several historical documents, since the beginning of the 15th century.