Publications

4528

HighlightsA portable system based on real-time fluorescence analysis was developed for field detection of Candidatus Liberibacter asiaticus within 40 min from sample to answer.A smartphone-assisted device was designed for easy operation, reliable nucleic acid amplification, and highly sensitive fluorescence detection, with sensitivity comparable to that of a commercial instrument.A novel homemade 3D printed box was used for in-field reagent storage, and it could maintain low temperature (<4°C) for about 8 hours without power supply.This fully integrated system is stable, easy to use, inexpensive, and has great application prospects in resource-limited areas.Abstract. Candidatus Liberibacter asiaticus (Las) is a main causal agent of huanglongbing (HLB), a destructive disease that has greatly reduced citrus yields and quality. Instruments with high sensitivity and portability are urgently required for on-site testing. In this study, a novel sample-to-answer optical system for on-site detection of Las was developed. Three major functions, including DNA extraction, amplification, and detection, are integrated into a portable case. This system mainly consists of (1) a specially designed 3D printed box for on-site reagent storage that can maintain low temperature (below 4°C) for 7.5 h at ambient temperature (35°C); (2) a custom device, called the IF-Device, for DNA amplification and detection of HLB, with an optimized optical structure, a sensitive signal processing circuit, and a precise temperature control algorithm with an accuracy of ±0.1°C; and (3) a battery-based power supply for the whole system. In a typical test using sodium fluorescein as a standard model, the results showed that the sensitivity of this system (1.0 nM) could easily meet the requirements of fluorescence biosensors. The feasibility of this homemade system was evaluated with samples extracted from infected citrus leaves based on the loop-mediated isothermal amplification (LAMP) method, and the limit of detection (LOD) was approximately 1.0 × 10-4 ng µL-1. The whole detection process for eight samples could be simultaneously accomplished within 40 min, and the results could be displayed on a smartphone in real-time. Moreover, the portable case is anti-interference, low cost, and only 2 kg in weight. Considering its sensitivity, stability, and portability, this highly integrated system possesses promising prospects for in-field detection. Keywords: Field detection, Fluorescence biosensor, Huanglongbing, Isothermal amplification, Sample-to-answer.

Sugarcane Grassy Shoot (SCGS) disease is known to be related to Rice Yellow Dwarf (RYD) phytoplasmas (16SrXI-B group) which are found predominantly in sugarcane growing areas of the Indian subcontinent and South-East Asia. The 16S rRNA gene sequences of SCGS phytoplasma strains belonging to the 16SrXI-B group share 98.07 % similarity with ‘Ca. Phytoplasma cynodontis’ strain BGWL-C1 followed by 97.65 % similarity with ‘Ca. P. oryzae’ strain RYD-J. Being placed distinctly away from both the phylogenetically related species, the taxonomic identity of SCGS phytoplasma is unclear and confusing. We attempted to resolve the phylogenetic positions of SCGS phytoplasma based on the phylogenetic analysis of 16S rRNA gene (>1500 bp), nine housekeeping genes (>3500 aa), core genome phylogeny (>10 000 aa) and OGRI values. The draft genome sequences of SCGS phytoplasma (strain SCGS) and Bermuda Grass White leaf (BGWL) phytoplasma (strain LW01), closely related to ‘Ca. P. cynodontis’, were obtained. The SCGS genome was comprised of 29 scaffolds corresponding to 505 173 bp while LW01 assembly contained 21 scaffolds corresponding to 483 935 bp with the fold coverages over 330× and completeness over 90 % for both the genomes. The G+C content of SCGS was 19.86 % while that of LW01 was 20.46 %. The orthoANI values for the strain SCGS against strains LW01 was 79.42 %, and dDDH values were 22. Overall analysis reveals that SCGS phytoplasma forms a distant clade in RYD group of phytoplasmas. Based on phylogenetic analyses and OGRI values obtained from the genome sequences, a novel taxon ‘Candidatus Phytoplasma sacchari’ is proposed.

‘Candidatus Liberibacter asiaticus’, an unculturable α-proteobacterium, is associated with citrus huanglongbing (HLB), a devastating disease threatening citrus production in Brazil and worldwide. In this study, a draft whole-genome sequence of ‘Ca. L. asiaticus’ strain 9PA from a sweet orange (cultivar Pera) tree collected in São Paulo State, Brazil, is reported. The 9PA genome is 1,231,881 bp, including two prophages, with G+C content of 36.7%. This is the first report of a whole-genome sequence of ‘Ca. L. asiaticus’ from Brazil or South America. The 9PA genome sequence will enrich ‘Ca. L. asiaticus’ genome resources and facilitate HLB research and control in Brazil and the world.

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

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

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.