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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: 218; Downloads: 111
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Abstract: The connected domination game is a variant of the domination game where the played vertices must form a connected subgraph at all stages of the game. In this paper we prove that deciding whether the game connected domination number is smaller than a given integer is PSPACE-complete using log-space reductions for both Dominator- and Staller-start connected domination game.
Keywords: connected domination game, complexity, PSPACE-complete
Published in DiRROS: 06.10.2025; Views: 240; Downloads: 100
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Published in DiRROS: 06.10.2025; Views: 300; Downloads: 171
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Keywords: high-entropy alloy, laser cladding, rare earth
Published in DiRROS: 03.10.2025; Views: 306; Downloads: 147
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Abstract: Mutual-visibility sets were motivated by visibility in distributed systems and social networks, and intertwine with several classical mathematical areas. Monotone properties of the variety of mutual-visibility sets, and restrictions of such sets to convex and isometric subgraphs are studied. Dual mutual-visibility sets are shown to be intrinsically different from other types of mutual-visibility sets. It is proved that for every finite subset $Z$ of positive integers there exists a graph $G$ that has a dual mutual-visibility set of size ▫$i$▫ if and only if $i\in Z\cup \{0\}$, while for the other types of mutual-visibility such a set consists of consecutive integers. Visibility polynomials are introduced and their properties derived. As a surprise, every polynomial with nonnegative integer coefficients and with a constant term $1$ is a dual visibility polynomial of some graph. Characterizations are given for total mutual-visibility sets, for graphs with total mutual-visibility number $1$, and for sets which are not total mutual-visibility sets, yet every proper subset is such. Along the way an earlier result from the literature is corrected.
Keywords: mutual-visibility sets, variety of mutual-visibility sets, convex subgraphs, integer polynomial
Published in DiRROS: 03.10.2025; Views: 257; Downloads: 135
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