First report of apple hammerhead viroid infecting apple trees in Montenegro.

Apple hammerhead viroid (AHVd, Pelamoviroid, Avsunviroidae) is one of the five viroids infecting apples. It has been identified on all continents except Australia since its viroid nature was confirmed (DiSerio et al. 2018; CABI and EPPO 2022). AHVd has been found in apple trees showing leaf mosaic, ringspot and dieback (Hamdi et al., 2021). Apple (Malus domestica Borkh.) and its wild relatives are traditionally grown in Montenegro. With an annual production of 7767 tons on 216 ha, it is the second most important fruit tree (after plum) in the country (Anonymous 2022). In a 2020-2022 survey, 29 apple trees exhibiting virus-like symptoms (e.g. mosaic, necrosis) were sampled throughout Montenegro, including 16 locations in eight municipalities (Podgorica, Danilovgrad, Niksic, Mojkovac, Bijelo Polje, Berane, Pljevlja and Savnik). Small RNAs were isolated using the mirVana miRNA Isolation Kit (Ambion, Life Technologies) and pooled into three bulk samples. Each bulk contained 9 to 10 samples. Libraries of sRNAs were constructed using the Ion Total RNA-Seq Kit v2 and barcoded using the Xpress RNA-Seq Barcode 1-16 Kit (Ion Torrent) according to the manufacturer's instructions. Small RNA library sequencing was performed on Illumina platform (Novogene Europe) yielding 9.9, 9.8 and 18.6 million reads in the three libraries. The CLC Genomics Workbench software was used to demultiplex the reads into pools using the 'Demultiplex Reads' tool. The online program VirusDetect (Zheng et al. 2017) was used for virus/viroid detection and identification. Besides viruses known to infect apple (apple stem grooving virus, apple stem pitting virus, apple mosaic virus), contigs mapping to AHVd were identified in all three bulks enabling full AHVd genomes reconstruction. To verify AHVd presence, all 29 apple samples were tested by reverse transcription-polymerase chain reaction (RT-PCR) using the AHVd PG13f/PG12r primers (Messmer et al. 2017). AHVd amplicons were obtained in three samples (30/21, 32/21 and 38/21) from bulk 1 and two samples (47/21 and 55/21) from bulk 2, while all samples from bulk 3 tested negative potentially due to the low titer of the pathogen or nucleotide mismatches at the 3' end of the primers. The three amplicons from bulk 1 were Sanger sequenced and partial AHVd genomes over 200 nts were obtained from two of them (30/21 and 32/21) (GenBank acc. nos. OQ863319 and OR020603). Furthermore, three full consensus AHVd genomes were assembled in Geneious Prime by mapping Sanger sequences onto contigs from Virus Detect and named 30/21, 32/21 and 38/21 (acc. nos. PP133245, -46, and -47, respectively). All three genomes exhibited conserved hammerhead motifs (Messmer et al. 2017). In BLASTn analysis, the isolate 30/21 from Montenegro shared the highest nt identity (98.8%) with the isolate SA-36 (ON564299) from Czechia, while 32/21 and 38/21 showed the highest identities (95.4% and 92.3%) with isolates SD17_2-3 (MK188691) from Canada and JF2 (ON564298) from Czechia, respectively. To the best of our knowledge, this is the first report of AHVd infecting Malus domestica in Montenegro. The AHVd-positive samples 30/21 and 32/21 originated from at least two-decade-old apple trees from Niksic, whilst 38/21 came from a 40-year-old tree from Mojkovac district, suggesting that this viroid has long been present in different parts of the country. The AHVd discovery in Montenegro should be considered in any phytosanitary regulations and pome fruit certification program in the country.

Increased Diversity of Citrus Tristeza Virus in Europe.

This study investigated the genetic diversity of citrus tristeza virus (CTV) isolates from Montenegro and Croatia, European countries with the northernmost citrus growing regions situated on the Eastern Adriatic coast. Fifteen complete or nearly complete CTV genomes were reconstructed from high-throughput sequencing of samples collected in distinct municipalities in Montenegro and Opuzen municipality in Croatia. Phylogenetic analyses assigned some of the sequences to VT and T30 strains, previously recorded in Europe, while remarkably other isolates were placed in S1 and RB groups, which have not been reported in Europe so far. In addition, a new phylogenetic lineage including only isolates from Montenegro was delineated and tentatively proposed as the MNE cluster. Recombination analysis revealed evidence of 11 recombination events in the sequences obtained in this study, between isolates of related strains, within isolates of the same strain, and between distant strains. These findings show that CTV diversity in Europe is higher than reported before and calls for the re-evaluation of management strategies.

Clematis vitalba 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.

First report of potato virus S and potato virus Y in tomatoes from Croatia.

Potato virus Y (PVY, genus Potyvirus) is an economically important aphid-transmissible virus with a very wide host range reported in many tomato-growing areas (Rivarez et al. 2021). Potato virus S (PVS, genus Carlavirus) has a limited host range (Lin et al. 2014) and occurs in tomato (Predajna et al. 2017), mostly in mixed infections with other viruses. In 2021, greenhouse tomatoes from Vidovec (46° 17' 3.4'' N, 16° 15' 37.0'' E) in the northwestern and Sedlarica (45° 54' 23.0'' N, 17° 12' 0.5'' E) in the eastern regions of Croatia were surveyed for virus-like diseases. In total, 30 plants were sampled (12 from Vidovec and 18 from Sedlarica) showing symptoms of mild mottling, leaf rugosity and mild bronzing followed by leaf necroses later in the season. Nucleic acids were extracted from leaves by adapted CTAB procedure (Murray and Thompson 1980) and DNase treated. Four representative samples from Vidovec and four from Sedlarica were pooled for high throughput sequencing (HTS). After rRNA depletion (RiboMinus™ Plant Kit for RNA-Seq, Invitrogen) and polyA tailing, two location specific libraries (PCR-cDNA sequencing kit, Oxford Nanopore Technologies) were prepared for nanopore HTS on MinION Mk1C device. From Vidovec samples, 459,285 raw reads (mean length 354 nt) were obtained and 206,718 (mean length 446 nt) from Sedlarica and mapped (Minimap2, v.2.17) against Kraken2 viral genome sequences database (https://benlangmead.github.io/aws-indexes/k2). The number of reads mapped to PVS genome was 1004 from Vidovec (coverage depth 1.56) and those mapped to PVY genome were 781 (coverage depth 0.99) and 57 (coverage depth 1), from Vidovec and Sedlarica, respectively. The PVS complete consensus genome from Vidovec (ON468562, 8485 nt) had 99.09% nucleotide identity (BLASTn) to a potato isolate from the Netherlands (MF418030). The PVY consensus genome sequences from Vidovec (ON505007, 9698 nt) and Sedlarica (ON505008, 9698 nt) had respectively 98.37% and 98.48% identities to a tomato isolate from Slovakia (MW685827). Reverse transcription polymerase chain reaction (RT-PCR) was performed for all 30 samples and amplicons were Sanger sequenced, with primers PVS-7773F/PVS-3'endR for a 720 nt PVS genome portion spanning the 3'-part of the CP and a complete 11K gene (Lin et al. 2014) and PVY-2F/2R primers for a 510 nt portion of PVY CP gene (Aramburu et al. 2006). Only one tomato out of 12 ('Borana') from Vidovec harbored PVS in the mixed infection with PVY. Two additional tomatoes from Vidovec and two from Sedlarica were infected solely by PVY. Amplicon sequences of PVS (ON651427) and PVY (ON707000-4, ON734067-8) had 100% identity with the HTS assembled sequences. The PVS isolate from Croatia grouped with PVSO (ordinary) strain in phylogenetic analysis and the PVY isolates from both sites grouped with the PVY-NTN strain (Cox and Jones 2010). Although PVY is considered to be widespread in tomato (Nikolic et al. 2018; Rivarez et al. 2021), this is its first report from Croatia. PVS, newly reported from Croatia here, is probably not associated with the symptoms recorded because the same symptomatology was observed in the singly and mixed infected 'Borana' tomato plants. The occurrence of PVY in the geographically distant (100 km apart) Vidovec and Sedlarica, suggests that it is widespread in the continental Croatia where tomatoes are commercially grown in plastic greenhouses. Further analyses are needed to elucidate PVY and PVS epidemiology and impact on the local tomato production.

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.

Genomic analysis of the brassica pathogen turnip mosaic potyvirus reveals its spread along the former trade routes of the Silk Road.

Plant pathogens have agricultural impacts on a global scale and resolving the timing and route of their spread can aid crop protection and inform control strategies. However, the evolutionary and phylogeographic history of plant pathogens in Eurasia remains largely unknown because of the difficulties in sampling across such a large landmass. Here, we show that turnip mosaic potyvirus (TuMV), a significant pathogen of brassica crops, spread from west to east across Eurasia from about the 17th century CE. We used a Bayesian phylogenetic approach to analyze 579 whole genome sequences and up to 713 partial sequences of TuMV, including 122 previously unknown genome sequences from isolates that we collected over the past five decades. Our phylogeographic and molecular clock analyses showed that TuMV isolates of the Asian-Brassica/Raphanus (BR) and basal-BR groups and world-Brassica3 (B3) subgroup spread from the center of emergence to the rest of Eurasia in relation to the host plants grown in each country. The migration pathways of TuMV have retraced some of the major historical trade arteries in Eurasia, a network that formed the Silk Road, and the regional variation of the virus is partly characterized by different type patterns of recombinants. Our study presents a complex and detailed picture of the timescale and major transmission routes of an important plant pathogen.

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.

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.

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 (24.5%) and wild boars (12.3%), 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.

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.

Single-strand conformation polymorphism analysis for differentiating phytoplasma strains.

Single-strand conformation polymorphism (SSCP) analysis is a sensitive and rapid technique for detecting DNA polymorphisms and mutations in PCR-amplified fragments. Due to its technical simplicity, it is widely used as a screening tool in various investigations, ranging from clinical diagnosis of human hereditary diseases to the characterization of microbial communities. This method can also be used successfully on phytoplasmas as a tool for the detection of molecular variability in conserved housekeeping genes such as 16S rRNA and tuf, as well as in more variable genes, revealing the presence of polymorphisms undetected by routine RFLP analyses. The reliability of SSCP has been confirmed by multiple alignments and phylogenetic analyses of representative sequences showing different SSCP profiles. However, it is not broadly applied in phytoplasma research yet. The technique provides an inexpensive, convenient, and sensitive method for determining sequence variation and to differentiate phytoplasma strains, and is particularly suitable for epidemiological studies or as a fast screening, typing tool when dealing with a large number of field samples.

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 68 % according to species. Percentage of substitution varied between 9 and 12 % for aceF, whereas it was between 5 and 6 % 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.

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.

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.

Purification of plant viral and satellite double-stranded RNAs on DEAE monoliths.

Replicative double-stranded RNA (dsRNA) is useful in preliminary identification of Cucumber mosaic virus and its satellite RNA (satRNA). This plant pathogen complex yields sufficient quantity of the replicative RNA form that can be isolated by chromatography on chemically unmodified graded cellulose powder (CF-11). In this work, much faster and more efficient procedure using DEAE monoliths was developed in which dsRNA was separated from other species in total nucleic acids extract originating from the infected plant tissue. The developed chromatographic method revealed the pathogens' presence in only 15 min, avoiding nucleic acid precipitation and electrophoretic analysis.