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Abstract: 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.
Keywords: plant pathogen, nanopore, grapevine, enrichment, HTS, Flavescence dorée, phytoplasma
Published in DiRROS: 08.10.2025; Views: 145; Downloads: 69
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Abstract: 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.
Keywords: WCLaV-1, WCLaV-2, watermelon, viruses, diagnostics
Published in DiRROS: 22.04.2025; Views: 664; Downloads: 295
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Abstract: Detection of begomoviruses capable of infecting tomatoes and plants of the family Cucurbitaceae.
Keywords: begomoviruses, EURL-Virology
Published in DiRROS: 02.09.2024; Views: 777; Downloads: 597
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Abstract: Phytoplasmas of the 16SrIII group are wide spread, and have a broad plant host range. Among these, ‘Candidatus phytoplasma pruni’ (‘Ca. P. pruni’; phytoplasmas of 16SrIII subgroup A) can cause serious diseases in Prunus species and ‘Ca. P. pruni’-related strains can infect other plant species, including grapevines. In this study, a new real-time PCR detection system was developed for ‘Ca. P. pruni’ using TaqMan chemistry. This test was designed to detect ‘Ca. P. pruni’, by amplifying the species-specific secY gene. In addition, a test to amplify the group-specific 16S rRNA gene region was also developed. The performances of both tests were evaluated. The test that amplifies the secY gene provided reliable and quick detection of ‘Ca. P. pruni’. Using the newly developed and validated test, ‘Ca. P. pruni’ was not found in any of the 434 field samples collected from different plants species grown in different regions of Slovenia.
Keywords: phytoplasma, X-disease, real-time PCR, Prunus
Published in DiRROS: 22.07.2024; Views: 1012; Downloads: 538
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Abstract: 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.
Keywords: plant viruses, plant virus detection, plant virology, high-throughput sequencing, RNA sequencing, plant tissues, plant pathogen, diagnostics, high-throughput sequencing, metagenomics, metatranscriptomics
Published in DiRROS: 12.07.2024; Views: 1233; Downloads: 400
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