Darko Vončina

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.

Viral diversity in autochthonous croatian grapevine cultivars

A survey was conducted on nine autochthonous grapevine cultivars grown along the Croatian coastal region. In total, 48 vines (44 from germplasm collection, 4 from vineyards) originating from 23 sites were tested for 26 viruses using molecular methods. Results revealed high infection rates with Grapevine leafroll-associated virus 3 (GLRaV-3); Grapevine virus A (GVA, both 91.7%); Grapevine fleck virus (GFkV, 87.5%); and Grapevine rupestris stem pitting-associated virus (GRSPaV, 83.3%). Other detected viruses were: Grapevine fanleaf virus (GFLV); Grapevine leafroll-associated viruses 1, 2, and strains of 4 (GLRaV-1, GLRaV-2, GLRaV-4); Grapevine viruses B, D, F (GVB, GVD, GVF); Grapevine red globe virus (GRGV); Grapevine vein feathering virus (GVFV); Grapevine Syrah virus 1 (GSyV-1); and Grapevine Pinot gris virus (GPGV). No virus-free vine was found. Mixed infections were determined in all vines, the number of viruses in a single vine ranged from three to nine. GLRaV-3 variant typing confirmed presence of group I, II, and III. Four vines with leaf deformation and mottling were positive for GPGV. Seven viruses (GLRaV-4-like group, GVD, GVE, GVF, GRGV, GSyV-1, and GVFV) were detected for the first time in Croatia. This survey confirmed the deteriorated sanitary status of autochthonous Croatian grapevine cultivars.

Infection and Colonization of Nicotiana benthamiana by Grapevine leafroll-associated virus 3

Grapevine leafroll disease is an increasing problem in all grape-growing regions of the world. The most widespread agent of the disease, Grapevine leafroll-associated virus 3 (GLRaV-3), has never been shown to infect species outside of the genus Vitis. Virus transmission to several plant species used as model systems was tested using the vine mealybug, Planococcus ficus. We show that GLRaV-3 is able to infect Nicotiana benthamiana. Working with GLRaV-3 infected N. benthamiana revealed distinct advantages in comparison with its natural host Vitis vinifera, yielding both higher viral protein and virion concentrations in western blot and transmission electron microscopy observations, respectively. Immunogold labelling of thin sections through N. benthamiana petioles revealed filamentous particles in the phloem cells of GLRaV-3 positive plants. Comparison of assembled whole genomes from GLRaV-3 infected V. vinifera vs. N. benthamiana revealed substitutions in the 5 UTR. These results open new avenues and opportunities for GLRaV-3 research.

Screening of some Croatian autochthonous grapevine varieties reveals a multitude of viruses, including novel ones

Next-generation sequencing of total RNA samples from four Croatian autochthonous grapevine varieties revealed the presence of a novel virus in two grapevine accessions. The complete genome sequence of a novel virus, tentatively named “grapevine badnavirus 1” (GBV-1), was reconstructed from a de novo-assembled contig. GBV-1 has a genome of 7,145 nucleotides containing three ORFs with sequence similarity to other badnaviruses. In addition, several other viruses and viroids, including grapevine virus G, grapevine virus K/D, grapevine virus T, grapevine Roditis leaf discoloration-associated virus, grapevine yellow speckle viroids 1 and 2, and hop stunt viroid were detected and identified for the first time in Croatian grapevines in the course of this study.

FIRST REPORT OF FIG MOSAIC VIRUS AND FIG BADNAVIRUS 1 ON COMMON FIG TREES IN CROATIA

Common fig (Ficus carica L.) is one of the most common fruit trees in the Croatian coastal region. Fig mosaic symp- toms, first reported more than a half century ago (Perisic , 1952) are found on most cultivars. In 2014, leaf samples were taken from 30 autochthonous fig trees from two locations of the Istrian peninsula for laboratory analyses. Twenty six trees exhibited typical symptoms of foliar mosaic, one tree exhibited also fasciations, while the remaining four trees were asymptomatic. Extraction of total RNA was done us- ing an RNeasy plant mini kit (Qiagen, Germany) and ex- traction of DNA using a GenElute Plant Genomic DNA Miniprep Kit (Sigma-Aldrich, USA). Plants were tested by PCR for the presence of Fig mosaic virus (FMV) and Fig badnavirus 1 (FBV-1), applying previously described condi- tions and diagnostic primers (Elbeaino et al., 2009; Laney et al., 2012). A symptomless fig tree was used as negative control. The infection with FMV was confirmed in 26 symp- tomatic out of 30 sampled trees (87%), while all 30 plants were positive for FBV-1. One amplicon each of FMV and FBV-1 (isolates FMV-Cro-19S and FBV-1-Cro-17S, respec- tively), were sequenced in both directions (GenBank acces- sion Nos KT312843 and KT312844). A BLASTN program search showed 97% identity at the nucleotide level of the FMV-Cro-19S RdRp gene with the comparable sequence of FMV SB2-2 isolate from Serbia (AB697838), while the poly- protein gene from FBV-1-Cro-17S had a sequence identical to that of several FBV-1 isolates from the USA (JN050864, JN050875, JN112365). To the best of our knowledge, this is the first report of FMV and FBV-1 in Croatia.

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

FIRST REPORT OF LITTLE CHERRY VIRUS 2 ON PRUNUS CERASUS var. MARASCA IN CROATIA

Little cherry virus 2 (LChV-2) is considered an important pathogen of cherries and, as such, testing of plant material is required according to the EPPO certification scheme PM 4/29 for cherries. In Croatia sour cherry Marasca is a well-known native variety with total production of 1500-1800 tons of fresh fruit per year, used mainly for production of renowned liqueurs and juices. In June 2014, 19 trees were selected and total RNA was extracted from leaves with the RNeasy plant mini kit (Qiagen, Germany). RT-PCR for LChV-2 was performed using two sets of primers: LCHV2LO2/LCHV2UP2 (Rott and Jelkmann, 2001), amplifying a 438 bp fragment of the methyltransferase (MT) gene, and LC26L/LC26R (Eastwell and Bernardy, 2001), amplifying a 409 bp fragment of the RdRp gene. Six trees were positive, four with the first primer set and two with the second. However, none of the trees tested positive by both sets, suggesting the presence of significant sequence variability among LChV-2 isolates (Theilmann et al., 2004) and the presence of at least two virus variants in Croatia. One PCR product for each primer set was sequenced from both directions and sequences were compiled using MEGA6 software. BLAST searches indicated that the part of the MT gene of Croatian isolate M-79 (GenBank accession No. KT369315) shares 88% identity with the USA6b isolate of LChV-2 (AF531505), while isolate M-75 (KT369316) was closest to Canadian strain LC5 (AF416335), sharing 96% identity with part of the RdRp gene. During the 2015 harvest period, some of the LChV-2-infected trees displayed the characteristic symptoms of uneven ripening. To the best of our knowledge this is the first report of LChV-2 in Croatia.

INCIDENCE OF VIRUSES ON AUTOCHTHONOUS AND INTRODUCED OLIVE VARIETIES IN CROATIAN ISTRIA DETECTED BY THREE DIAGNOSTIC TECHNIQUES

A survey on viruses infecting olive trees was performed in Croatian Istria. Major olive viruses were detected using molecular (RT-PCR), biological (bioassay on indicator plants) and serological (DAS-ELISA) diagnostic techniques. Specifically, fifteen olive varieties from a total of 62 olive trees have been tested for the presence of eight viruses. Cherry leaf roll virus (CLRV), Strawberry latent ring spot virus (SLRSV) and Olive latent virus-1 (OLV-1) were detected by one-step RT-PCR in ten autochthonous and five introduced olive varieties. The most frequent found was CLRV, detected in 11.3% of tested trees both in autochthonous and in introduced varieties, while OLV-1 and SLRSV were detected only in autochthonous varieties and in lower incidence. In order to confirm the results obtained by one-step RT-PCR, inocula prepared from the flowers were used for inoculation of herbaceous indicator plants. Symptoms on herbaceous hosts were monitored and CLRV and SLRSV were detected by DAS-ELISA, confirming the results of molecular test. The most successful mechanical transmission was on Chenopodium quinoa, while the most evident symptoms were observed on Cucumis sativus L. cv. Suncani potok plants.

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.

Virus composition influences virus elimination success and in vitro growth characteristics of the Grapevine cv. Plavac mali

Shoot tip cultures coupled with thermotherapy was used for the production of virus- free candidate clones of the Croatian autochthonous and most important red-berried grapevine cultivar Plavac mali. The procedure was successful for the elimination of Grapevine leafroll- associated virus 3 (GLRaV-3) and Grapevine fleck virus (GFkV), but not Grapevine leafroll- associated virus 1 (GLRaV-1). Results showed that a selective virus eradication occurred depending on the initial composition of the viral population of treated samples. When the in vitro growth of cv. Plavac mali explants harbouring different viruses was compared, it was found that, similarly to virus elimination, tissue proliferation was virus composition- dependent. This is a first report on virus elimination in cv. Plavac mali and, by and large, from grapevines in Croatia.