1. 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: 116; Prenosov: 48
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2. 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: 119; Prenosov: 42
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4. In-depth study of tomato and weed viromes reveals undiscovered plant virus diversity in an agroecosystemMark Paul Selda Rivarez, Anja Pecman, Katarina Bačnik, Olivera Maksimović, Ana Vučurović, Gabrijel Seljak, Nataša Mehle, Ion Gutiérrez-Aguirre, Maja Ravnikar, Denis Kutnjak, 2023, izvirni znanstveni članek Povzetek: Background: In agroecosystems, viruses are well known to influence crop health and some cause phytosanitary and economic problems, but their diversity in non-crop plants and role outside the disease perspective is less known. Extensive virome explorations that include both crop and diverse weed plants are therefore needed to better understand roles of viruses in agroecosystems. Such unbiased exploration is available through viromics, which could generate biological and ecological insights from immense high-throughput sequencing (HTS) data. Results: Here, we implemented HTS-based viromics to explore viral diversity in tomatoes and weeds in farming areas at a nation-wide scale. We detected 125 viruses, including 79 novel species, wherein 65 were found exclusively in weeds. This spanned 21 higher-level plant virus taxa dominated by Potyviridae, Rhabdoviridae, and Tombusviridae, and four non-plant virus families. We detected viruses of non-plant hosts and viroid-like sequences and demonstrated infectivity of a novel tobamovirus in plants of Solanaceae family. Diversities of predominant tomato viruses were variable, in some cases, comparable to that of global isolates of the same species. We phylogenetically classified novel viruses and showed links between a subgroup of phylogenetically related rhabdoviruses to their taxonomically related host plants. Ten classified viruses detected in tomatoes were also detected in weeds, which might indicate possible role of weeds as their reservoirs and that these viruses could be exchanged between the two compartments. Conclusions: We showed that even in relatively well studied agroecosystems, such as tomato farms, a large part of very diverse plant viromes can still be unknown and is mostly present in understudied non-crop plants. The overlapping presence of viruses in tomatoes and weeds implicate possible presence of virus reservoir and possible exchange between the weed and crop compartments, which may influence weed management decisions. The observed variability and widespread presence of predominant tomato viruses and the infectivity of a novel tobamovirus in solanaceous plants, provided foundation for further investigation of virus disease dynamics and their effect on tomato health. The extensive insights we generated from such in-depth agroecosystem virome exploration will be valuable in anticipating possible emergences of plant virus diseases and would serve as baseline for further post-discovery characterization studies.
Ključne besede: tomato, weed, virus, viroid, virome, virus discovery, virus diversity, phylogenetics, metagenomics, viromics
Objavljeno v DiRROS: 13.04.2023; Ogledov: 589; Prenosov: 128
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5. Chloroplast redox state changes mark cell-to-cell signaling in the hypersensitive responseTjaša Lukan, Anže Županič, Tjaša Mahkovec Povalej, Jacob O. Brunkard, Mirjam Kmetič, Mojca Juteršek, Špela Baebler, Kristina Gruden, 2023, izvirni znanstveni članek Povzetek: Bisphenol A (BPA) is one of the most commonly used substances in the manufacture ofvarious everyday products. Growing concerns about its hazardous properties, including endocrinedisruption and genotoxicity, have led to its gradual replacement by presumably safer analogues inmanufacturing plastics. The widespread use of BPA and, more recently, its analogues has increasedtheir residues in the environment. However, our knowledge of their toxicological profiles is limitedand their combined effects are unknown. In the present study, we investigated the toxic effectscaused by single bisphenols and by the combined exposure of BPA and its two analogues, BPAP andBPC, after short (24-h) and prolonged (96-h) exposure in HepG2 spheroids. The results showed thatBPA did not reduce cell viability in HepG2 spheroids after 24-h exposure. In contrast, BPAP andBPC affected cell viability in HepG2 spheroids. Both binary mixtures (BPA/BPAP and BPA/BPC)decreased cell viability in a dose-dependent manner, but the significant difference was only observedfor the combination of BPA/BPC (both at 40μM). After 96-h exposure, none of the BPs studiedaffected cell viability in HepG2 spheroids. Only the combination of BPA/BPAP decreased cellviability in a dose-dependent manner that was significant for the combination of 4μM BPA and 4μMBPAP. None of the BPs and their binary mixtures studied affected the surface area and growth ofspheroids as measured by planimetry. In addition, all BPs and their binary mixtures studied triggeredoxidative stress, as measured by the production of reactive oxygen species and malondialdehyde,at both exposure times. Overall, the results suggest that it is important to study the effects of BPsas single compounds. It is even more important to study the effects of combined exposures, as thecombined effects may differ from those induced by single compounds.
Ključne besede: chloroplast redox state, hypersensitive response (HR)-conferred resistance, immune signaling, live cell imaging, Solanum tuberosum (potato), spatiotemporal analysis, stromules, virus resistance
Objavljeno v DiRROS: 13.04.2023; Ogledov: 393; Prenosov: 126
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7. 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, izvirni znanstveni članek Ključne besede: grapevine, water status, virus infection, GFLV, xylem vessel occlusion, gene expression
Objavljeno v DiRROS: 06.09.2022; Ogledov: 694; Prenosov: 240
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