Publications

4795

"Candidatus Liberibacter solanacearum" is a gram-negative, fastidious bacterium that inhabits and multiplies in the phloem of host plants. The bacterium causes economically important diseases of solanaceous crops such as "zebra chips" of potatoes, and chlorotic weakening of cultivated plants of the Apiaceae family. Beside potatotes, this pathogen causes significant economic losses in tobacco, peper and tomato production. Insect vectors classified in the family Triozidae play an important role in the epidemiology of this bacterium. Insects transfer bacterial cells from infected to healthy plants by feeding on the plant juice from the phloem tissue. This pathogenic bacterium could be transmitted by infected seeds which result in long-distance spread. Based on the analysis of single nucleotide polymorphism (SNP), nine haplotypes (A, B, C, D, E, F, G, H, U) from different geographical regions, host plants and insect vectors have been described. Considering to impossibility of isolation of this bacterium on a nutrient medium, detection and identification are based on the application of molecular methods (conventional PCR, real-time PCR, multiplex PCR, nested PCR and LAMP PCR). Taking into consideration the increasing spread of "Candidatus Liberibacter solanacearum" in Europe, as well as its recent detection in Serbia, the aim of this paper was to raise an awareness about importance of this bacterium and summarize the protocols and molecular methods for its detection and identification.

Abstract Ther.mo.ha.lo.bac.te.ra.ce'ae. N.L. masc. n. Thermohalobacter , type genus of the family; suff. ‐aceae , ending to denote a family; N.L. fem. pl. n. Thermohalobacteraceae , the Thermohalobacter family. Free‐living bacteria that mainly occur in anoxic saline environments. Cells are not pigmented and have a Gram‐positive type of cell wall. Gram‐stain reaction can be negative or positive. Typically slender rod‐shaped cells that are motile by flagella. Several strains form heat‐resistant spores. Mainly euryhaline, moderately thermophilic and halophilic. Members of the family are strictly anaerobic bacteria that utilize carbohydrates or peptides by fermentation. Cellular fatty acid patterns are dominated by iso ‐C 15:0 and iso ‐C 15:0 DMA. The family currently accommodates members of the genera Thermohalobacter , Caldisalinibacter , Caloranaerobacter , Brassicibacter , Sporosalibacterium , and Clostridiisalibacter , which were previously affiliated with the family Clostridiaceae . The affiliation of novel species to this family depends on the phylogenetic position, which should be determined on the basis of comparative sequence analyses of 16S rRNA genes or genomes. DNA G + C content (mol%): 28–33. Type genus: Thermohalobacter Cayol et al. 2000. Taxonomic and Nomenclature Notes According to the List of Prokaryotic names with Standing in Nomenclature (LPSN), the taxonomic status of the family Thermohalobacteraceae is: synonym (last update, February 2025) * . Correct name: Clostridiaceae LPSN classification: Bacteria / Bacillati / Bacillota / Clostridia / Eubacteriales / Clostridiaceae The family Thermohalobacteraceae can also be recovered in the Genome Taxonomy Database (GTDB) as f__Thermohalobacteraceae (version v220) ** . GTDB classification: d__Bacteria / p__Bacillota_A / c__Clostridia / o__Tissierellales / f__Thermohalobacteraceae * Meier‐Kolthoff et al. ( 2022 ). Nucleic Acids Res , 50 , D801 – D807 ; DOI: 10.1093/nar/gkab902 ** Parks et al. ( 2022 ). Nucleic Acids Res , 50 , D785 – D794 ; DOI: 10.1093/nar/gkab776

Abstract Phy.to.plas‐ma. Gr. masc. n. phytos a plant; N.L. neut. n. Phytoplasma a form from a plant. Phytoplasmas are wall‐less, nutritionally fastidious, and phytopathogenic prokaryotes 0.2–0.8 μm in diameter that morphologically resemble nonhelical members of the class Mollicutes . Sequencing of nearly full‐length PCR‐amplified 16S rRNA genes, combined with earlier studies, provided the first comprehensive phylogeny of the organisms and showed that they constitute a unique, monophyletic clade within the Mollicutes . These organisms are most closely related to members of the genus Acholeplasma within the Anaeroplasma clade as defined by Weisburg et al. Despite decades of efforts and recent progress, sustained pure culture in cell‐free media has not yet been demonstrated for any phytoplasma. Their genome sizes have been estimated to range from 530 to 1,350 kb, and the DNA G + C content is about 23–30 mol%. The presence of a characteristic oligonucleotide sequence in the 16S rRNA gene, CAA GAY BAT KAT GTK TAG CYG GDC T, and standard codon usage indicate that phytoplasmas represent a distinct genus‐level taxon for which the name ‘ Candidatus Phytoplasma’ has been adopted. At present, for any subtaxa within the provisional genus, the designation ‘ Candidatus ’ must still be used. DNA G + C content (mol%) : 23–29. Taxonomic and Nomenclature Notes According to the List of Prokaryotic names with Standing in Nomenclature (LPSN), the taxonomic status of the genus Candidatus Phytoplasma is: preferred name (not correct name) (last update, February 2025) * . LPSN classification: Bacteria / Bacillati / Mycoplasmatota / Mollicutes / Acholeplasmatales / Acholeplasmataceae / Candidatus Phytoplasma Candidatus Phytoplasma could not be recovered in GTDB ** . * Meier‐Kolthoff et al. ( 2022 ). Nucleic Acids Res , 50 , D801 – D807 ; DOI: 10.1093/nar/gkab902 ** Parks et al. ( 2022 ). Nucleic Acids Res , 50 , D785 – D794 ; DOI: 10.1093/nar/gkab776

Tick-borne agents constitute a growing concern for human and animal health worldwide. Hyalomma aegyptium is a hard tick with a three-host life cycle, whose main hosts for adults are Palearctic tortoises of genus Testudo. Nevertheless, immature ticks can feed on a variety of hosts, representing an important eco-epidemiological issue regarding H. aegyptium pathogens circulation. Hyalomma aegyptium ticks are vectors and/or reservoirs of various pathogenic agents, such as Ehrlichia, Anaplasma, Babesia and Hepatozoon/Hemolivia. Ehrlichia and Anaplasma are emergent tick-borne bacteria with a worldwide distribution and zoonotic potential, responsible for diseases that cause clinical manifestations that grade from acute febrile illness to a fulminant disease characterized by multi-organ system failure, depending on the species. Babesia and Hepatozoon/Hemolivia are tick-borne parasites with increasing importance in multiple species. Testudo graeca tortoises acquired in a large animal market in Doha, Qatar, were screened for a panel of tick-borne pathogens by conventional PCR followed by bidirectional sequencing. The most prevalent agent identified in ticks was Hemolivia mauritanica (28.6%), followed by Candidatus Midichloria mitochondrii (9.5%) and Ehrlichia spp. (4.7%). All samples were negative for Babesia spp. and Hepatozoon spp. Overall, 43% of the examined adult ticks were infected with at least one agent. Only 4.7% of the ticks appeared to be simultaneously infected with two agents, i.e., Ehrlichia spp. and H. mauritanica. This is the first detection of H. mauritanica, Ehrlichia spp. and Candidatus M. mitochondrii in H. aegyptium ticks collected from pet spur-thighed tortoises, in Qatar, a fact which adds to the geographical extension of these agents. The international trade of Testudo tortoises carrying ticks infected with pathogens of veterinary and medical importance deserves strict control, in order to reduce potential exotic diseases.

In Australia, Stylosanthes little leaf (StLL) phytoplasma has been detected in Stylosanthes scabra Vogel, Arachis pintoi Krapov, Saccharum officinarum L., Carica papaya L., Medicago sativa L., and Solanum tuberosum L. The 16S rRNA gene sequence of StLL phytoplasma strains from S. scabra, C. papaya, S. officinarum and S. tuberosum were compared and share 99.93–100 % nucleotide sequence identity. Phylogenetic comparisons between the 16S rRNA genes of StLL phytoplasma and other ‘Candidatus Phytoplasma’ species indicate that StLL represents a distinct phytoplasma lineage. It shares its most recent known ancestry with ‘Ca. Phytoplasma luffae’ (16SrVIII-A), with which it has 97.17–97.25 % nucleotide identity. In silico RFLP analysis of the 16S rRNA amplicon using iPhyClassifier indicate that StLL phytoplasmas have a unique pattern (similarity coefficient below 0.85) that is most similar to that of ‘Ca. Phytoplasma luffae’. The unique in silico RFLP patterns were confirmed in vitro. Nucleotide sequences of genes that are more variable than the 16S rRNA gene, namely tuf (tu-elongation factor), secA (partial translocation gene), and the partial ribosomal protein (rp) gene operon (rps19-rpl22-rps3), produced phylogenetic trees with similar branching patterns to the 16S rRNA gene tree. Sequence comparisons between the StLL 16S rRNA spacer region confirmed previous reports of rrn interoperon sequence heterogeneity for StLL, where the spacer region of rrnB encodes a complete tRNA-Isoleucine gene and the rrnA spacer region does not. Together these results suggest that the Australian phytoplasma, StLL, is unique according to the International Organization for Mycoplasmology (IRPCM) recommendations. The novel taxon ‘Ca. Phytoplasma stylosanthis’ is proposed, with the most recent strain from a potato crop in Victoria, Australia, serving as the reference strain (deposited in the Victorian Plant Pathology Herbarium as VPRI 43683).

Microbiology is in a new age in which sequence databases are primary sources of information about many microbes. However, in-depth analysis of environmental genomes thus retrieved is essential to substantiate the new knowledge.