Dijana Škorić

Multilocus sequence analysis reveals the genetic diversity of European fruit tree phytoplasmas and supports the existence of inter-species recombination.

The genetic diversity of three temperate fruit tree phytoplasmas Candidatus Phytoplasma prunorum, Ca. P. mali and Ca. P. pyri has been established by multilocus sequence analysis. Among the four genetic loci used, the genes imp and aceF distinguished 30 and 24 genotypes, respectively, and showed the highest variability. Percentage of substitution for imp ranged from 50 to 68u200a% according to species. Percentage of substitution varied between 9 and 12u200a% for aceF, whereas it was between 5 and 6u200a% for pnp and secY. In the case of Ca P. prunorum the three most prevalent aceF genotypes were detected in both plants and insect vectors, confirming that the prevalent isolates are propagated by insects. The four isolates known to be hypo-virulent had the same aceF sequence, indicating a possible monophyletic origin. Haplotype network reconstructed by eBURST revealed that among the 34 haplotypes of Ca. P. prunorum, the four hypo-virulent isolates also grouped together in the same clade. Genotyping of some Spanish and Azerbaijanese Ca. P. pyri isolates showed that they shared some alleles with Ca. P. prunorum, supporting for the first time to our knowledge, the existence of inter-species recombination between these two species.

A framework for the evaluation of biosecurity, commercial, regulatory and scientific impacts of plant viruses and viroids identified by NGS technologies

Recent advances in high-throughput sequencing technologies and bioinformatics have generated huge new opportunities for discovering and diagnosing plant viruses and viroids. Plant virology has undoubtedly benefited from these new methodologies, but at the same time, faces now substantial bottlenecks, namely the biological characterization of the newly discovered viruses and the analysis of their impact at the biosecurity, commercial, regulatory, and scientific levels. This paper proposes a scaled and progressive scientific framework for efficient biological characterization and risk assessment when a previously known or a new plant virus is detected by next generation sequencing (NGS) technologies. Four case studies are also presented to illustrate the need for such a framework, and to discuss the scenarios.

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.

The use of SSCP analysis in the assessment of phytoplasma gene variability

Single-strand conformation polymorphism (SSCP) analysis is a broadly used technique for detecting mutations. The aim of this work was to assess the applicability of SSCP as a new tool for the detection of the molecular variability of uncultivable mollicutes - phytoplasmas. Three phytoplasma regions were investigated: 16S rDNA, tuf gene, and dnaB gene. Fragments amplified by PCR were subjected to SSCP under conditions optimized for each fragment length. In all of the analyzed regions, SSCP revealed the presence of polymorphism undetected by routine RFLP analyses. Reliability of the method was confirmed by the multiple alignments and phylogenetic analyses of representative sequences showing different SSCP profiles.

East Adriatic—a reservoir region of severe Citrus tristeza virus strains

Citrus tristeza virus (CTV) represents one of the major threats to citrus production worldwide. In the East Adriatic region, CTV symptoms are mostly absent due to traditional citrus grafting on trifoliate orange (Poncirus trifoliata), a CTV-tolerant rootstock. Therefore, the virus has been continuously spreading by the propagation of infected material. The genetic variability of CTV was studied on nineteen citrus samples, collected from orchards in the coastal region of Croatia, Montenegro and Albania, that previously tested positive by ELISA and immunocapture RT-PCR. Single-strand conformation polymorphism of the amplified coat protein gene demonstrated the presence of different CTV variants in each amplicon, while sequence analysis of cloned CP gene variants confirmed their clustering into six out of the seven phylogenetic groups so far delineated. Four of these groups include sequences of severe quick decline, seedling yellows and stem-pitting (SP) isolates, thought to be found only rarely in the Mediterranean region. Regardless of the lack of symptoms in the field, CTV isolates from the East Adriatic displayed high genetic variability and pathogenic potential, additionally confirmed by biological characterisation. The high percentage of mixed infections suggest the potential for further diversification and a greater risk of severe variants spreading into new areas.

Distribution and molecular characterization of Hepatitis E virus in domestic animals and wildlife in Croatia

Hepatitis E is becoming a growing health concern in European countries as an increase of sporadic human cases of unknown origin has been recorded lately. Its causative agent, Hepatitis E virus (HEV), is known to have zoonotic potential and thus the role of domestic and wild animals in the chain of viral spread should be considered when investigating risk factors and the epidemiology of the disease. A comprehensive survey based on viral RNA detection was carried out in Croatia including blood, spleen and liver samples originating from 1816 different domestic and wild animals and digestive gland samples from 538 molluscs. A high HEV prevalence was detected in domestic pigs (24.5xa0%) and wild boars (12.3xa0%), whereas cattle, molluscs, ruminant and carnivore wildlife samples tested negative. Molecular characterization of both ORF1 and ORF2 genomic regions confirmed the phylogenetic clustering of the obtained sequences into genotype 3, previously reported in Europe. Furthermore, our results proved the presence of identical sequence variants in different samples, regardless of their origin, age or habitat of the host, suggesting transmission events between domestic swine, as well as between domestic swine and wild boars in the country. Moreover, a close genetic relationship of Croatian animal strains and known human HEV strains from GenBank opens the question of possible cross-species HEV transmission in Croatia, especially in the areas with an intensive swine production.

Occurrence of Stem-Pitting Strains of Citrus tristeza virus in Croatia

Citrus is grown in Croatia (approximately 1,500 ha of citrus groves) on the Dalmatian Coast and Islands between 42 and 43°30N. The major species, Citrus unshiu Marc. (Satsuma mandarin), is grafted on trifoliate rootstock. The presence of Citrus tristeza virus (CTV) in Satsumas in the Neretva Valley Region was previously reported (3). During the course of a biomolecular characterization of isolates from Croatia, 15 budsticks were collected from field-infected, enzyme-linked immunosorbent assay (ELISA)-positive sources during the autumn of 2003 near Kaštela, Split, Metković (Neretva Valley), and on the island of Vis. Isolates were propagated by graft transmission to Madam Vinous sweet orange (SwO) and maintained in an insect-proof greenhouse at 21 to 33°C. Eight months later, the bark of terminal twigs was peeled off, and the wood was examined for the occurrence of pits. Typical tristeza stem-pitting (SP) was observed in four isolates originating from cvs. Fukumoto navel, Washington navel, and Ichimaru Satsuma and C. wilsonii. The bark from the infected sources was analyzed using immunocapture reverse transcription-polymerase chain reaction (RT-PCR) with primers CTV1 and CTV10 (1), targeting the whole coat protein (CP) gene. The PCR products of the expected size (669 nucleotides) were obtained and TA cloned (pGEM-T Easy Vector; Promega, Madison, WI) in E. coli cells. Thirty-two clones harboring the CTV CP gene were sequenced. Two of the SP isolates contained four genomic variants that differed an average of 2.0% from the severe SwO SP strains SY568 and Nuaga (4) from California and Japan, respectively. The other two SP isolates contained four variants that differed as little as 1.6% from the severe SwO SP from India, CTV-Puna, and CTV-Bangalore (2). The net average distance between these two clusters of sequences is 5.2%. One sequence from each of the four SP isolates was deposited in GenBank (Accession Nos. AY791841 to AY791844). These findings were confirmed by direct observation of SP symptoms in a Satsuma orchard in the Neretva Valley during the spring of 2004. No other conspicuous symptoms that could be attributed to CTV were observed in the field. Most Satsumas were introduced to the Neretva Region from Japan between 1964 and 1984. Together with the fact that the related Nuaga strain was also isolated from Satsumas in Japan (4), our results suggest that SwO SP strains were introduced into Croatia at the same time and have been spreading for several decades. It has been generally believed that this kind of CTV strains either do not exist in the Mediterranean basin or, when found (e.g., Spain), are immediately eradicated. The findings reported here suggest that the epidemiological scenario for the Mediterranean Basin requires revision. References: (1) G. Nolasco et al. Eur. J. Plant Pathol. 108:293, 2002. (2) A. Roy et al. Arch. Virol. 148:707, 2003. (3) A. Šarić and I. Dulić. Agric. Conspectus Sci. 55:171, 1990. (4) G. Suastika et al. J. Gen. Plant Pathol. 67:73, 2001.

A case study of FD and BN phytoplasma variability in Croatia: multigene sequence analysis approach

Uncultivable bacteria from the genus ‘Candidatus Phytoplasma’ are associated with grapevine yellows (GY) diseases worldwide. In Euro-Mediterranean viticultural areas, GY are most frequently caused by Bois Noir (BN) and Flavescence Dorée (FD) phytoplasmas. Surveys of GY in Croatia have been conducted regularly since 1997. BN phytoplasmas have been found to be widespread, while FD phytoplasmas were recently discovered in restricted areas of the country. The aim of this study was to assess the variability of genotypes involved in GY pathosystems by a multilocus sequence typing (MLST) approach. Grapevine, weed and insect vector samples were collected from three locations. Species-specific stamp and vmp1 genes, together with house-keeping genes tuf and secY, were amplified from BN strains. In FD strains, the genes secY, map and uvrB-degV were analyzed. MLST revealed a diversity of BN genotypes, one of which was prevalent and identified in samples of grapevine and the insect vector Hyalesthes obsoletus, corroborating their affiliation to the same pathosystem. Distinct BN strains found in bindweed and two grapevine samples indicated the presence of different BN pathosystems involving a yet unidentified vector, possibly from the genus Reptalus. Moreover, a co-occurence of BN and FD phytoplasma in the same vineyard was identified. The genotyping of FD strains from both grapevine and Scaphoideus titanus showed the presence of at least two distinct FD genotypes at two different locations, suggesting separate introductions of the disease into the country. In this study, MLST proved to be a useful and informative tool in advancing the understanding of GY epidemiology in Croatia.

A novel application of methacrylate based short monolithic columns: Concentrating Potato spindle tuber viroid from water samples☆

Potato spindle tuber viroid (PSTVd) is the causal agent of a number of agriculturally important diseases. It is a single-stranded, circular and unencapsidated RNA molecule with only 356-360 nucleotides and no coding capacity. Because of its peculiar structural features, it is very stable ex vivo and it is easily transmitted mechanically by contaminated hands, tools, machinery, etc. In this work, we describe the development and optimization of a method for concentrating PSTVd using Convective Interaction Media (CIM) monolithic columns. The ion-exchange chromatography on diethylamine (DEAE) monolithic analytical column (CIMac DEAE-0.1 mL) resulted in up to 30% PSTVd recovery whilst the hydrophobic interaction chromatography on C4 monolithic analytical column (CIMac C4-0.1 mL) improved it up to 60%. This was due to the fact that the binding of the viroid to the C4 matrix was less strong than to the highly charged anion-exchange matrix and could be easier and more completely eluted under the applied chromatographic conditions. Based on these preliminary results, a C4 HLD-1 (High Ligand Density) 1 mL monolithic tube column was selected for further experiments. One-litre-water samples were mixed with different viroid quantities and loaded onto the column. By using reverse transcription quantitative polymerase chain reaction (RT-qPCR), the viroid RNA was quantified in the elution fraction (≈5 mL) indicating that 70% of the viroid was recovered and concentrated by at least two orders of magnitude. This approach will be helpful in screening irrigation waters and/or hydroponic systems nutrient solutions for the presence of even extremely low concentrations of PSTVd.

Extensively and multi drug-resistant Acinetobacter baumannii recovered from technosol at a dump site in Croatia

In a karst pit above City of Rijeka in Croatia the hazardous industrial waste was continuously disposed from 1955 to 1990, and later it was periodically used as an illegal dump site. The surface part of a technosol at the edge of dump was analysed mineralogically, geochemically and bacteriologically. From the technosol rich in petroleum hydrocarbons and heavy metals three isolates of Acinetobacter baumannii were recovered. Isolates from technosol shared many features that are previously described for clinically isolates: the affiliation to IC1 and 2, multi-drug resistant (MDR) or extensively drug-resistant (XDR) antibiotic resistance profile, carbapenem resistance mediated by blaOXA72 and blaOXA23 genes, and the expression of virulence factors. In in vitro conditions, isolates were able to survive in contact with technosol during 58days of monitoring. The most probable source of A. baumannii in technosol was the illegally disposed hospital waste. Proper management and disposal of human solid waste is mandatory to prevent the spread of clinically important A. baumannii in nature.