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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: 922; Downloads: 469
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Abstract: Physiological studies in plants often require enzyme extraction from tissues containing high concentrations of phenolsand polyphenols. Unless removed or neutralized, such compounds may hinder extraction, inactivate enzymes, and inter-fere with enzyme detection. The following protocol for activity assays for enzymes of primary carbohydrate metabo-lism, while based on our recently published one for quantitative measurement of activities using coupled spectrophoto-metric assays in a 96-well format, is tailored to the complexities of phenolic- and anthocyanin-rich extracts from grape-vine leaf. As a case study we applied the protocol to grapevine leaf samples infected with plant pathogenic bacteriašCandidatusPhytoplasma solani’, known to alter carbohydrate metabolism in grapevine. The described adaptations maybe useful for determination of metabolic fingerprints for physiological phenotyping of other plant species with inhe-rently high levels of phenolic compounds.
Keywords: AGPase, carbohydrates, invertases, sucrose synthase, panel of enzyme activity assays, phytoplasma
Published in DiRROS: 25.07.2024; Views: 1380; Downloads: 766
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Abstract: Phytoplasmas from the 16Sr-X apple proliferation (AP) group are quarantine species in Europe and causal agents of the most important diseases of fruit trees within the family Rosaceae, namely apple proliferation, European stone fruit yellows and pear decline. In this study, a detailed insight into the molecular diversity of isolates of two phytoplasmas from the AP group, i. e. ‘Candidatus Phytoplasma mali’ and ‘Ca. P. prunorum’ obtained from different orchards in Slovenia, was estimated by a multilocus sequence typing, based on analysis of the genomic regions of aceF, pnp, secY and imp. With seven and five genotypes defined for ‘Ca. P. mali’ and ‘Ca. P. prunorum’ isolates, respectively, imp was the most variable among the applied markers. On the other hand, pnp was the least variable with three genotypes defined for ‘Ca. P. mali’ isolates and only one for ‘Ca. P. prunorum’ isolates. The presented results complete the survey of the AP group phytoplasma diversity in Slovenia, which has started with the recent analysis of the ‘Ca. P. pyri’. The comparison of results with those from several European countries shows an important genetic diversity of the Slovenian genotypes with some previously unknown. The genotype distribution reflects the geographic position of Slovenia. Additional grafting experiments with apricot trees tolerant to ‘Ca. P. prunorum’ demonstrated that the tolerance status is transmissible. Some possible mechanisms involved in the process are discussed.
Keywords: apple proliferation, multilocus sequence typing, phytoplasma diversity, Slovenia
Published in DiRROS: 24.07.2024; Views: 1374; Downloads: 894
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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: 1651; Downloads: 893
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