31. The physiological impact of GFLV virus infection on grapevine water status : first observationsAnastazija Jež Krebelj, Maja Cigoj, Marija Stele, Marko Chersicola, Maruša Pompe Novak, Paolo Sivilotti, 2022, izvirni znanstveni članek Povzetek: In a vineyard, grapevines are simultaneously exposed to combinations of several abiotic (drought, extreme temperatures, salinity) and biotic stresses (phytoplasmas, viruses, bacteria). With climate change, the incidences of drought in vine growing regions are increased and the host range of pathogens with increased chances of virulent strain development has expanded. Therefore, we studied the impact of the combination of abiotic (drought) and biotic (Grapevine fanleaf virus (GFLV) infection) stress on physiological and molecular responses on the grapevine of cv. Schioppettino by studying the influence of drought and GFLV infection on plant water status of grapevines, on grapevine xylem vessel occlusion, and on expression patterns of 9-cis-epoxycarotenoid dioxygenase 1 (NCED1), 9-cis-epoxycarotenoid dioxygenase 2 (NCED2), WRKY encoding transcription factor (WRKY54) and RD22-like protein (RD22) genes in grapevines. A complex response of grapevine to the combination of drought and GFLV infection was shown, including priming in the case of grapevine water status, net effect in the case of area of occluded vessels in xylem, and different types of interaction of both stresses in the case of expression of four abscisic acid-related genes. Our results showed that mild (but not severe) water stress can be better sustained by GFLV infection rather than by healthy vines. GFLV proved to improve the resilience of the plants to water stress, which is an important outcome to cope with the challenges of global warming. Ključne besede: grapevine, water status, virus infection, GFLV, xylem vessel occlusion, gene expression Objavljeno v DiRROS: 16.07.2024; Ogledov: 96; Prenosov: 105
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32. Hydrodynamic cavitation efficiently inactivates potato virus Y in waterArijana Filipić, Tadeja Lukežič, Katarina Bačnik, Maja Ravnikar, Meta Ješelnik, Tamara Košir, Martin Petkovšek, Mojca Zupanc, Matevž Dular, Ion Gutiérrez-Aguirre, 2022, izvirni znanstveni članek Povzetek: Waterborne plant viruses can destroy entire crops, leading not only to high financial losses but also to food shortages. Potato virus Y (PVY) is the most important potato viral pathogen that can also affect other valuable crops. Recently, it has been confirmed that this virus is capable of infecting host plants via water, emphasizing the relevance of using proper strategies to treat recycled water in order to prevent the spread of the infectious agents. Emerging environmentally friendly methods such as hydrodynamic cavitation (HC) provide a great alternative for treating recycled water used for irrigation. In the experiments conducted in this study, laboratory HC based on Venturi constriction with a sample volume of 1 L was used to treat water samples spiked with purified PVY virions. The ability of the virus to infect plants was abolished after 500 HC passes, corresponding to 50 min of treatment under pressure difference of 7 bar. In some cases, shorter treatments of 125 or 250 passes were also sufficient for virus inactivation. The HC treatment disrupted the integrity of viral particles, which also led to a minor damage of viral RNA. Reactive species, including singlet oxygen, hydroxyl radicals, and hydrogen peroxide, were not primarily responsible for PVY inactivation during HC treatment, suggesting that mechanical effects are likely the driving force of virus inactivation. This pioneering study, the first to investigate eukaryotic virus inactivation by HC, will inspire additional research in this field enabling further improvement of HC as a water decontamination technology. Ključne besede: hydrodynamic cavitation, potato virus Y, virus inactivation, water decontamination Objavljeno v DiRROS: 16.07.2024; Ogledov: 128; Prenosov: 115
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33. Diversity and pathobiology of an ilarvirus unexpectedly detected in diverse plants and global sequencing dataMark Paul Selda Rivarez, Chantal Faure, Laurence Svanella-Dumas Svanella-Dumas, Anja Pecman, Magda Tušek-Žnidarič, Deborah Schönegger, Maja Ravnikar, Denis Kutnjak, 2023, izvirni znanstveni članek Ključne besede: phylogenetics, histopathology, pollen, virus diversity, virion morphology, symptomatology, virus transmission Objavljeno v DiRROS: 12.07.2024; Ogledov: 103; Prenosov: 112
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34. Managing the deluge of newly discovered plant viruses and viroids : an optimized scientific and regulatory framework for their characterization and risk analysisNuria Fontdevila Pareta, Maryam Khalili, Ayoub Maachi, Mark Paul Selda Rivarez, Johan Rollin, Ferran Salavert Pamblanco, Coline Temple, Miguel A. Aranda, Denis Kutnjak, Maja Ravnikar, 2023, izvirni znanstveni članek Povzetek: The advances in high-throughput sequencing (HTS) technologies and bioinformatic tools have provided new opportunities for virus and viroid discovery and diagnostics. Hence, new sequences of viral origin are being discovered and published at a previously unseen rate. Therefore, a collective effort was undertaken to write and propose a framework for prioritizing the biological characterization steps needed after discovering a new plant virus to evaluate its impact at different levels. Even though the proposed approach was widely used, a revision of these guidelines was prepared to consider virus discovery and characterization trends and integrate novel approaches and tools recently published or under development. This updated framework is more adapted to the current rate of virus discovery and provides an improved prioritization for filling knowledge and data gaps. It consists of four distinct steps adapted to include a multi-stakeholder feedback loop. Key improvements include better prioritization and organization of the various steps, earlier data sharing among researchers and involved stakeholders, public database screening, and exploitation of genomic information to predict biological properties. Ključne besede: plant viruses and viroids, high throughput sequencing (HTS), biological characterization, plant health, regulatory agencies, Pest Risk Analysis (PRA), virus disease Objavljeno v DiRROS: 12.07.2024; Ogledov: 133; Prenosov: 81
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35. Tomato brown rugose fruit virus in aqueous environments : survival and significance of water-mediated transmissionNataša Mehle, Katarina Bačnik, Irena Bajde, Jakob Brodarič, Adrian Fox, Ion Gutiérrez-Aguirre, Miha Kitek, Denis Kutnjak, Yue Lin Loh, Olivera Maksimović, Maja Ravnikar, Elise Vogel, Christine Vos, Ana Vučurović, 2023, izvirni znanstveni članek Povzetek: Tomato brown rugose fruit virus (ToBRFV) has recently emerged as a major disease of tomatoes and peppers. ToBRFV is a seed- and contact-transmitted virus. In Slovenia, ToBRFV RNA was detected in samples of wastewater, river, and water used to irrigate plants. Even though the source of detected RNA could not be clearly established, this raised the question of the significance of the detection of ToBRFV in water samples and experimental studies were performed to address this question. The data presented here confirm that the release of virus particles from the roots of infected plants is a source of infectious ToBRFV particles in water and that the virus can remain infective up to four weeks in water stored at room temperature, while its RNA can be detected for much longer. These data also indicate that irrigation with ToBRFV-contaminated water can lead to plant infection. In addition, it has been shown that ToBRFV circulated in drain water in commercial tomato greenhouses from other European countries and that an outbreak of ToBRFV can be detected by regular monitoring of drain water. A simple method for concentrating ToBRFV from water samples and a comparison of the sensitivity of different methods, including the determination of the highest ToBRFV dilution still capable of infecting test plants, were also investigated. The results of our studies fill the knowledge gaps in the epidemiology and diagnosis of ToBRFV, by studying the role of water-mediated transmission, and provide a reliable risk assessment to identify critical points for monitoring and control. Ključne besede: tomato brown rugose fruit virus, tomato, hydroponics, water-linked epidemiology, survival Objavljeno v DiRROS: 12.07.2024; Ogledov: 143; Prenosov: 149
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36. Looking beyond virus detection in RNA sequencing data : lessons learned from a community-based effort to detect cellular plant pathogens and pestsAnnelies Haegeman, Yoika Foucart, Kris De Jonghe, Thomas Goedefroit, Maher Al Rwahnih, Neil Boonham, Thierry Candresse, Yahya Gaafar, Oscar Hurtado-Gonzales, Zala Kogej Zwitter, Denis Kutnjak, Janja Lamovšek, Irena Mavrič Pleško, 2023, izvirni znanstveni članek Povzetek: High-throughput sequencing (HTS), more specifically RNA sequencing of plant tissues, has become an indispensable tool for plant virologists to detect and identify plant viruses. During the data analysis step, plant virologists typically compare the obtained sequences to reference virus databases. In this way, they are neglecting sequences without homologies to viruses, which usually represent the majority of sequencing reads. We hypothesized that traces of other pathogens might be detected in this unused sequence data. In the present study, our goal was to investigate whether total RNA-seq data, as generated for plant virus detection, is also suitable for the detection of other plant pathogens and pests. As proof of concept, we first analyzed RNA-seq datasets of plant materials with confirmed infections by cellular pathogens in order to check whether these non-viral pathogens could be easily detected in the data. Next, we set up a community effort to re-analyze existing Illumina RNA-seq datasets used for virus detection to check for the potential presence of non-viral pathogens or pests. In total, 101 datasets from 15 participants derived from 51 different plant species were re-analyzed, of which 37 were selected for subsequent in-depth analyses. In 29 of the 37 selected samples (78%), we found convincing traces of non-viral plant pathogens or pests. The organisms most frequently detected in this way were fungi (15/37 datasets), followed by insects (13/37) and mites (9/37). The presence of some of the detected pathogens was confirmed by independent (q)PCRs analyses. After communicating the results, 6 out of the 15 participants indicated that they were unaware of the possible presence of these pathogens in their sample(s). All participants indicated that they would broaden the scope of their bioinformatic analyses in future studies and thus check for the presence of non-viral pathogens. In conclusion, we show that it is possible to detect non-viral pathogens or pests from total RNA-seq datasets, in this case primarily fungi, insects, and mites. With this study, we hope to raise awareness among plant virologists that their data might be useful for fellow plant pathologists in other disciplines (mycology, entomology, bacteriology) as well. Ključne besede: plant viruses, plant virus detection, plant virology, high-throughput sequencing, RNA sequencing, plant tissues, plant pathogen, diagnostics, high-throughput sequencing, metagenomics, metatranscriptomics Objavljeno v DiRROS: 12.07.2024; Ogledov: 138; Prenosov: 112
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37. On stifling a transcendental breath : an Italian contribution to the philosophy of breathingMichael Lewis, 2023, izvirni znanstveni članek Povzetek: The article contends that respiratory philosophy has, thus far, laid predominant stress upon the empirical form of breathing, as opposed to the transcendental; or at least it has used breath precisely as an occasion to elide or deconstruct this very opposition. Breath is then conceived primarily as material, bodily, and natural: as binding us together with the animals and with all living things.and yet this apparently benign ecological gesture is not without its deleterious side-effects: by contrasting this gesture with a more humanistic and transcendental conception of breath, inspired by Giorgio agamben’s work on the voice, we might begin to gain some clarity as to the jarring contrast that sprang up between the friendly valorisation of a shared con-spiration that has characterised this young philosophy up to now, and the intense, even violent, hostility to the breath of the other which the developed world exhibited from at least 2020 to 2022.We consider whether an overly empiricistic conception of breath and of the human might have played a part in this reversal of values. In conclusion, the article urges upon us a certain turn towards the transcendental form of the breath, and indeed to a certain human exceptionalism in this regard. Ključne besede: Agamben, language, voice, breath, animal, human, humanism, transcendental, empirical, masks, stifling, identity, invisibility, virus, pandemic Objavljeno v DiRROS: 14.05.2024; Ogledov: 283; Prenosov: 229
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38. Fast and accurate multiplex identification and quantification of seven genetically modified soybean lines using six-color digital PCRAlexandra Bogožalec Košir, Sabine Muller, Jana Žel, Mojca Milavec, Allison C. Mallory, David Dobnik, 2023, izvirni znanstveni članek Povzetek: The proliferation of genetically modified organisms (GMOs) presents challenges to GMO testing laboratories and policymakers. Traditional methods, like quantitative real-time PCR (qPCR), face limitations in quantifying the increasing number of GMOs in a single sample. Digital PCR (dPCR), specifically multiplexing, offers a solution by enabling simultaneous quantification of multiple GMO targets. This study explores the use of the Naica six-color Crystal dPCR platform for quantifying five GM soybean lines within a single six-plex assay. Two four-color assays were also developed for added flexibility. These assays demonstrated high specificity, sensitivity (limit of detection or LOD < 25 copies per reaction) and precision (bias to an estimated copy number concentration <15%). Additionally, two approaches for the optimization of data analysis were implemented. By applying a limit-of-blank (LOB) correction, the limit of quantification (LOQ) and LOD could be more precisely determined. Pooling of reactions additionally lowered the LOD, with a two- to eight-fold increase in sensitivity. Real-life samples from routine testing were used to confirm the assays’ applicability for quantifying GM soybean lines in complex samples. This study showcases the potential of the six-color Crystal dPCR platform to revolutionize GMO testing, facilitating comprehensive analysis of GMOs in complex samples. Ključne besede: digital PCR, dPCR, quantification, multiplexing, genetically modified organisms, 6-color system, virus diagnostics, virology Objavljeno v DiRROS: 29.03.2024; Ogledov: 372; Prenosov: 163
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39. Increased diversity of citrus tristeza virus in EuropeJelena Zindović, Miroslav Čizmović, Ana Vučurović, Paolo Margaria, Dijana Škorić, 2023, izvirni znanstveni članek Povzetek: 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. Ključne besede: complete genomes, genotyping, citrus tristeza virus, CTV, non-EU strain Objavljeno v DiRROS: 29.03.2024; Ogledov: 427; Prenosov: 179
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