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Povzetek: Phytoplasmas are non-culturable obligate intracellular bacteria that cause considerable economic losses in agriculture. Genome sequencing provides crucial insights into their biology and vector dependence. However, genome studies on phytoplasmas are often hampered by their low abundance in naturally infected plants. Propagation in test plants is usually necessary but time-consuming and resource-intensive, especially for quarantine phytoplasmas such as the phytoplasma causing Flavescence dorée (FD), a serious threat to European viticulture. To overcome these challenges, we aimed to develop a protocol for efficient enrichment of phytoplasma DNA directly from field-collected samples, enabling genome sequencing using both Illumina and Oxford Nanopore Technologies platforms. We evaluated six sample preparation protocols that included stepwise enrichment steps to improve phytoplasma genome coverage and assembly quality. The most effective approach combined differential centrifugation, CTAB extraction and removal of CpG-methylated host DNA and resulted in a notable increase in the relative abundance of phytoplasma reads compared to other protocols. Rarefaction analysis of the dataset generated using this protocol demonstrated that the entire phytoplasma genome was covered by reads in a dataset comprising 3 billion nucleotides. We also evaluated and compared de novo phytoplasma genome assemblies generated from short Illumina reads and long nanopore sequencing reads. While Illumina sequencing yielded more accurate assemblies with longer total lengths, the assemblies derived from nanopore sequencing data contained longer individual contigs. This advantage was reflected in hybrid assemblies that combined both technologies, yielding longer phytoplasma contigs than assemblies from Illumina datasets and lower mismatch rates compared to assemblies from nanopore sequencing datasets. A hybrid de novo assembled genome of the Slovenian FD phytoplasma isolate achieved 96% reference genome coverage, with high contiguity and low error rates. This streamlined and accessible protocol enables high-quality genome sequencing of phytoplasma-infected grapevines without the need for propagation in test plants. This facilitates broader phytoplasma research and can potentially be extended to other naturally infected phytoplasma hosts or organisms infected with other non-culturable microbes.
Ključne besede: plant pathogen, nanopore, grapevine, enrichment, HTS, Flavescence dorée, phytoplasma
Objavljeno v DiRROS: 08.10.2025; Ogledov: 192; Prenosov: 91
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Ključne besede: begomoviruses, EURL-Virology
Objavljeno v DiRROS: 03.10.2025; Ogledov: 264; Prenosov: 68
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Povzetek: Tomato spotted wilt orthotospovirus (TSWV) is a major plant pathogen causing significant economic losses in tomato production worldwide. Understanding its genetic diversity and evolutionary mechanisms is crucial for effective disease management. This study analyzed TSWV isolates from symptomatic tomato plants collected across Croatia, Montenegro and Slovenia between 2020 and 2024. High-throughput sequencing (HTS) was employed to obtain whole-genome sequences, followed by phylogenetic analyses to assess genetic variability and relationships among isolates from these three countries and other isolates of worldwide geographic origin. Phylogenetic analyses placed all studied isolates within the L1-M3-S3 genotype, commonly associated with solanaceous crops in Europe. While Croatian and Slovenian isolates exhibited high genetic similarity, Montenegrin isolates clustered in a distinct subgroup, showing closer relationships to Asian and Mediterranean accessions. Despite the severe disease symptoms observed, no substitutions in the NSm protein associated with resistance-breaking (RB) phenotypes were detected. These findings suggest that additional virome components, environmental factors or so far unknown mechanism(s) may contribute to infection and disease severity in tomato and strongly support the need of continuous surveillance of TSWV genetic diversity in order to inform breeding programs and develop sustainable management strategies to mitigate future outbreaks.
Ključne besede: tomato, HTS, phylogeny, TSWV, plant virus
Objavljeno v DiRROS: 16.09.2025; Ogledov: 288; Prenosov: 134
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Povzetek: Pepino mosaic virus (PepMV) is a plant pathogen causing significant economic losses in tomato production. Sensitive, reliable, and robust detection methods are crucial for containing the spread of PepMV and reducing its damaging effects. Digital PCR (dPCR) presents several advantages to conventional real-time quantitative PCR (qPCR), including absolute quantification ability, robust quantitative multiplexing capabilities, and straightforward result analysis. Furthermore, dPCR is especially suitable for analysis of complex samples due to its remarkable tolerance to PCR inhibitors, which makes it a promising method for plant virus genotyping. In this chapter, we present two protocols for PepMV genotyping and quantification using one-step reverse transcription digital PCR (RT-dPCR). The first protocol outlines four simplex assays, while the second describes two duplex assays for precise and comprehensive genotyping of PepMV variants.
Ključne besede: plant viruses, detection, quantification, Pepino mosaic virus, PepMV, digital PCR, dPCR, RT-dPCR, duplex
Objavljeno v DiRROS: 08.09.2025; Ogledov: 316; Prenosov: 39
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Povzetek: In July 2024, a pooled leaf sample (D760/24) was collected from several plants of three watermelon cultivars (Citrullus lanatus cvs. Crimson Sweet, Asahi Miyako Hybrid F1 and Top Gun) grown in an open field (approx. 0.5ha) in Dombrava, Slovenia. The plants which were included in the pooled sample showed virus-like symptoms, such as leaf mosaic, wilting and necrosis (eXtra Supplementary material Fig. S1). The disease incidence was estimated at 10%. DNA and RNA were extracted following Mehle et al. (2013) and RNeasy Plant Mini Kit (Qiagen, Germany) protocols, respectively. The sample was tested positive by reverse-transcription (RT)-PCR for watermelon crinkle leaf-associated virus 1 (WCLaV-1) and WCLaV-2 ( Hernandez et al. 2021) and negative for other viruses (details on viruses tested and primers used are available in eXtra Table S1). The obtained amplicons of expected sizes of WCLaV-1 and WCLaV-2 movement protein (MP) and RNA-dependent RNA polymerase (RdRp) genes (eXtra Fig S2) were then subjected to Sanger sequencing (Eurofins Genomics, Germany) and BLAST analysis. The MP (PQ570004, PQ570006) and the RdRp (PQ570005, PQ570007) sequences exhibited 100% identity with multiple accessions of WCLaV-1, such as PP792977 and PP792976, and WCLaV-2, such as LC636073 and LC636074. Illumina high-throughput sequencing (HTS, Novogene, Germany, NovaSeq X Plus, PE150) identified WCLaV-1 (PV012703-04) and WCLaV-2 (PV012705-06) reads, along with cucumis melo amalgavirus 1 (CmAV1, PV012707) and solanum nigrum ilarvirus 1 reads (insufficient reads to reconstruct genome segments, it may originate from pollen contamination of nearby infected plants in the field (Rivarez et al. 2023)). HTS data were analyzed in CLC Genomics Workbench v. 24 (Qiagen, USA) using the pipeline by (Pecman et al. 2022). Consensus genome sequences were reconstructed by iterative read mapping to the most similar reference sequence of the virus obtained from NCBI GenBank. To check for WCLaVs in watermelon seeds sold in Slovenia, we tested five seed samples from Sugar Baby, Crimstar F1, and Crimson Sweet (three lots) by RT-PCR. We also tested four leaf samples from plants grown from these seeds at 3-5 true leaves stage. Both viruses were found in all seed and leaf extracts. However, mechanical inoculations with the sap of two samples (plants grown from infected seed sample and sample D760/24) on several commonly used indicator plants including Chenopodium quinoa, Capsicum annuum, Nicotiana clevelandii, Nicotiana glutinosa, Nicotiana benthamiana, Nicotiana tabacum cv. White Burley, Nicotiana rustica, Datura stramonium, Cucurbita pepo cv. Bianca di Trieste, and Cucurbita maxima did not result in their infection. Retrospective analyses of our HTS data of two watermelon and 84 other cucurbits samples from previous years showed WCLaV-1 and WCLaV-2 reads in two pooled samples (containing equal amount of RNA of each sample): one from 2018 and another from 2019. RT-PCR confirmed the presence of WCLaVs only in watermelons. The pool from 2018 was sequenced at GATC (Germany, NovaSeq 6000 S2, PE 150) and from 2019 in-house using Oxford Nanopore Technologies (UK, MinION Mk1B device, SQK-PCS108, R9 flow cell). All HTS reads are deposited in the NCBI Short Reads Archive (PRJNA1202089). This is the first report of WCLaV-1 and WCLaV-2 in Slovenia and Europe, the two viruses which were included to the Alert list of the European and Mediterranean Plant Protection Organization, due to limited knowledge about their epidemiology (EPPO 2023). Further research is necessary to determine the incidence of these viruses in Europe, elucidate their epidemiology, symptoms association and their potential impact on the production of watermelons in the region.
Ključne besede: WCLaV-1, WCLaV-2, watermelon, viruses, diagnostics
Objavljeno v DiRROS: 22.04.2025; Ogledov: 703; Prenosov: 316
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