Citruses in Croatia – cultivation, major virus and viroid threats and challenges

Although Croatia is not often perceived as a citrus growing country, citrus species have been planted in the Croatian southern coastal part for centuries. Citrus had always been important as a source of vitamins and micronutrients for local consumption, but with the introduction of Satsuma mandarins (Citrus unshiu Marc.) citrus production started its commercial development. The Croatian coast is probably the northernmost commercial citrus growing area globally with several agroecological constraints influencing citrus production. However, Satsuma mandarins, the most cold-tolerant citrus of economic interest, are successfully cultivated resulting in an excellent quality of fruits that easily finds a market niche. Unfortunately, with the import of plant material in the last century, the simultaneous introduction of viral and subviral pathogens occurred. The most important are Citrus tristeza virus and Citrus exocortis viroid. As certain rootstock-scion combinations are tolerant to virus or viroid infections and display no obvious symptoms, these pathogens have been continuously spreading by plant propagation. Molecular and biological analyses revealed the existence of pathotypes not believed to be endemically present in the Mediterranean region. Their high intra-isolate genetic variability and frequent coinfections suggest that this geographical region presents a sort of a biological enclave of potentially threatening pathogens. Their transmission from this agroecological niche to areas where sensitive rootstock-scion combinations are common presents a serious risk to citrus production, especially when global warming and the resulting migration of efficient vector species are considered. Comprehensive pathogen monitoring is important for improving citrus production and the prevention of pathogen transmission

Molecular characterization of Coleus blumei viroids 1 and 3 in Plectranthus scutellarioides in Croatia

Asymptomatic Plectranthus scutellarioides (syn. Coleus blumei) plants of four different cultivars were investigated for the presence of Coleus blumei viroid (CbVd) species (genus Coleviroid, family Pospiviroidae). Cloned and sequenced amplicons obtained by generic coleviroid primers were analyzed for their primary and secondary structure. CbVd-1 was found in one of the coleus cultivars in a single infection and in another cultivar in a mixed infection with CbVd-3. Additionally, CbVd-3 was found in a third coleus cultivar in a single infection. In singly and doubly infected cultivars, both CbVd-1 and CbVd-3 variants displayed higher variability regarding both length and nucleotide composition in a doubly infected cultivar. Sequences from different cultivars showed different phylogenetic grouping suggesting distinct infection sources, probably introduced infected seeds. For all phylogenetic subgroups, characteristic nucleotide changes were identified and their influence on the secondary structure was predicted. The most noticeable structural changes were observed in nucleotide positions 25–26 in CbVd-1 and in the position 138 in CbVd-3 variants. This work includes the first report of coleviroids in Croatia, the second reported coleviroids sequences from Europe and the third report of CbVd-3 in the world enlarging the pool of knowledge on the variability and distribution of viroids in horticultural crops.This study was funded by the University of Zagreb grants to D. Škorić and S. Černi numbers: 20281214, 20282308 and 20283119.Peer reviewe

Geographical distribution of 'flavescence dorée' phytoplasmas in Croatian grapevines

Abstract Seven Croatian grapevine samples found positive for the presence of 'flavescence dorée'(FD) phytoplasmas in 2010 survey confirm the first findings of FD phytoplasma from previous year, as well as the existence of new grapevine yellows heavily affected narrow area west and southwest of Zagreb along the border with Slovenia. Two new locations 100 km east and 60 km north-east of Zagreb, respectively, were found to be affected, widening the potential FD-risk zone and placing the capital in its centre. Grapevine varieties infected with FD-related phytoplasmas encompassed widely known Pinot Noir and Riesling, but also indigenous red Plemenka Crvena and Ružica Crvena, as well as white variety Škrlet. Preliminary molecular characterization results suggest the variability of 16SrV-C identified phytoplasmas, especially from the locations west and southwest of Zagreb

First viruses infecting cockspur coral tree (Erythrina crista-galli L.): discovery of a putative novel capillovirus and a new host for the prune dwarf virus

peer reviewedA cockspur coral tree (Erythrina crista-galli, Fabaceae) from the collection of woody ornamentals of the Botanical Garden (Faculty of Science) in Zagreb showed conspicuous virus-like symptoms. The leaf tissue was analyzed by high throughput sequencing (HTS), revealing the presence of two viruses: prune dwarf virus (PDV, genus Ilarvirus) and an unknown virus belonging to the genus Capillovirus (family Betaflexiviridae). The complete sequence of PDV RNA3 of 2,129 nucleotides (nts) coding for the coat and movement proteins was obtained. The complete coding region spanning 6,483 nts was obtained for the capillovirus. It contained all expected open reading frames, and its maximum nucleotide identity was 42% with apple stem grooving capillovirus sequence (GenBank accession number LC143387). As this is far below the threshold for species delineation within the genus Capillovirus, we propose the name Erythrina capillovirus (ErCV) for this putatively new virus and a new virus species Capillovirus ErCV in the family Betaflexiviridae. RT-PCR confirms the presence of ErCV and PDV in symptomatic E. crista-galli, an unusual and exotic fabaceous host with no viruses recorded yet. Asymptomatic Erythrina plants were tested negative for the two viruses in RT-PCR, which, together with their presence in the symptomatic plant, suggests the capillovirus and/or PDV might play a role in eliciting observed symptoms and deserve further investigations

Legacy of Plant Virology in Croatia—From Virus Identification to Molecular Epidemiology, Evolution, Genomics and Beyond

This paper showcases the development of plant virology in Croatia at the University of Zagreb, Faculty of Science, from its beginning in the 1950s until today, more than 70 years later. The main achievements of the previous and current group members are highlighted according to various research topics and fields. Expectedly, some of those accomplishments remained within the field of plant virology, but others make part of a much-extended research spectrum exploring subviral pathogens, prokaryotic plant pathogens, fungi and their viruses, as well as their interactions within ecosystems. Thus, the legacy of plant virology in Croatia continues to contribute to the state of the art of microbiology far beyond virology. Research problems pertinent for directing the future research endeavors are also proposed in this review

<i>Clematis vitalba</i> Is a Natural Host of the Novel Ilarvirus, Prunus Virus I

Clematis vitalba L. is a climbing shrub and a pioneer plant in abandoned orchards or vineyards that are widespread in temperate climate zones. In past years, several viruses infecting the Clematis species have been identified, including different ilarviruses. Prunus virus I (PrVI) is a recently described ilarvirus, which has been shown to infect sweet cherries and peaches in Greece. Moreover, its presence has been detected in ornamental Clematis in Russia. In the present work, we analyzed the virome of wildly growing C. vitalba plants from Hungary, Slovakia and Croatia showing different kinds of symptoms using high-throughput sequencing (HTS) of small RNAs or ribodepleted RNAs. Applying HTS enabled us to identify the presence of PrVI in C. vitalba, and the bioinformatic analyses were further validated with RT-PCR using PrVI-specific primers and Sanger dideoxy sequencing. Nearly full genome sequences of all three viral RNAs of one Hungarian, two Slovak and one Croatian isolate were determined. Their phylogenetic analysis showed high similarity to each other and to other PrVI isolates described from Central Europe. As the sampled plants were co-infected with other viruses, it is not possible to determine a direct correlation between the infection with PrVI and the observed symptoms. Analyses of different Prunus species in stock collection showed infection of several peach and sweet cherry varieties in Hungary. Our results expand the knowledge on the natural host range of PrVI and highlight the necessity to evaluate alternative plant hosts (even non-Prunus) of PrVI and the role of the virus in the etiology of the potential diseases

Stem pitting and seedling yellows symptoms of Citrus tristeza virus infection may be determined by minor sequence variants

The isolates of Citrus tristeza virus (CTV), the most destructive viral pathogen of citrus, display a high level of variability. As a result of genetic bottleneck induced by the bud-inoculation of CTV-infected material, inoculated seedlings of Citrus wilsonii Tanaka displayed different symptoms. All successfully grafted plants showed severe symptoms of stem pitting and seedling yellows, while plants in which inoculated buds died displayed mild symptoms. Since complex CTV population structure was detected in the parental host, the aim of this work was to investigate how it changed after the virus transmission, and to correlate it with observed symptoms. The coat protein gene sequence of the predominant genotype was identical in parental and grafted plants and clustered to the phylogenetic group 5 encompassing severe reference isolates. In seedlings displaying severe symptoms, the low-frequency variants clustering to other phylogenetic groups were detected, as well. Indicator plants were inoculated with buds taken from unsuccessfully grafted C. wilsonii seedlings. Surprisingly, they displayed no severe symptoms despite the presence of phylogenetic group 5 genomic variants. The results suggest that the appearance of severe symptoms in this case is probably induced by a complex CTV population structure found in seedlings displaying severe symptoms, and not directly by the predominant genomic variant