Publications

4434

‘Candidatus Phytoplasma brasiliense’ (CPB) is a phytoplasma originally discovered in South America and is known to infect a wide variety of economically important crops. It is most prevalent in Hibiscus spp., where it causes witches broom symptoms, and papaya, where it causes bunchy top. Recently, CPB was documented for the first time in North America in a new host, globe sedge. In this study, two quantitative PCR assays are developed: one using high-resolution melt curve analysis (HRMA) based on the secA gene and the other a TaqMan assay based on the dnaK gene. The secA/HRMA and dnaK/TaqMan assay successfully amplified two of the three isolates of CPB. Both assays were screened against available isolates of 16SrI, 16SrII, and 16SrIV phytoplasmas. The secA/HRMA assay failed to amplify 16SrI and 16SrIV phytoplasmas but successfully amplified 16SrII phytoplasmas. The resulting melting point (Tm) products of CPB and 16SrII phytoplasmas displayed a difference of 0.5°C, easily distinguishing them by melt curves. The dnaK/TaqMan assay failed to amplify all non-CPB phytoplasma isolates in the study. The development of these assays provides a valuable tool that will significantly improve monitoring programs in Florida and will aid in developing a better fundamental understanding of the epidemiology of this phytoplasma.

AbstractCulture collections such as the Blue Biotechnology and Ecotoxicology Culture Collection (LEGE‐CC) hold approximately 1200 cyanobacterial strains and are critical community resources. However, many isolates in this and other collections have not been described with a polyphasic approach, and this limits further study. Here, we employed a polyphasic methodology that integrates 16S rRNA gene phylogenetic analyses, similarity (p‐distance), 16S‐23S ITS rRNA region secondary structures, morphological analyses, and habitat assessments to describe four novel cyanobacterial genera from the LEGE‐CC, Portugal. Pseudolimnococcus planktonicus gen. et sp. nov. (Chroococcales) is phylogenetically and morphologically related to Limnococcus. The 16S rRNA gene similarity between the types of both genera is only 93.1%. Morphologically, Pseudolimnococcus cells do not reach the original spherical shape before the next division or have aerotopes and firm mucilage, while Limnococcus cells reach the original shape, lack aerotopes, and have diffluent mucilage. Eucapsopsis lusitanus gen. et sp. nov. (Chroococcales) is morphologically similar to Eucapsis but differs from it by having aerotopes and diffluent envelope. Eucapsis lacks aerotopes and has firm mucilaginous envelopes, rarely diffluent. Both genera are phylogenetically very distant from each other and have only 90.68% 16S rRNA gene similarity. Pseudoacaryochloris arrabidensis gen. et sp. nov. (Acaryochloridales) differs from Acaryochloris by the lack of mucilaginous envelope, which is present in Acaryochloris. Both genera are phylogenetically distant and have only 94.1% 16S rRNA gene similarity. Moreover, Acaryochloris is marine (sponge symbiont), while Pseudoacaryochloris is from freshwater. Vasconcelosia minhoensis gen. et sp. nov. (Nodosilineales) is phylogenetically related to Cymatolege but has only 94.3% similarity with this genus. Morphologically both genera are distinct. Vasconcelosia has a Romeria‐like structure, while Cymatolege has a Phormidium‐like structure. In all cases the 16S‐23S ITS rRNA region secondary structures are in agreement with the other analyses. These novel genera expand the diversity of cyanobacteria in culture collections.

We investigated the rise of nontuberculous mycobacteria (NTM) infections in Bulgaria, focusing on species identification and distribution from 2018 to 2022. Utilizing advanced diagnostic tools, including the Hain Mycobacterium CM/AS method, Myco-biochip assay, and whole-genome sequencing, the study identifies and characterizes a diverse range of Mycobacterium species from clinical samples. While M. avium, M. gordonae, M. fortuitum, and M. chelonae were dominating, a number of rare species were also found. They include such species as M. marseillense and M. celatum. Moreover, the noticeable prevalence of M. terrae complex species missed by conventional testing was observed. We identified a rare species, highly homologous to previously described strains from Japan; based on genome–genome distance data, we propose its reannotation as a new species. Further, a novel species was identified, which is significantly distinct from its closest neighbor, M. iranicum, with ANI = 87.18%. Based on the SeqCode procedure, we propose to name this new species Mycobacterium bulgaricum sp. nov. Dynamic changes in NTM species prevalence in Bulgaria observed from 2011 to 2022 highlight the emergence of new species and variations tied to environmental and demographic factors. This underscores the importance of accurate species identification and genotyping for understanding NTM epidemiology, informing public health strategies, and enhancing diagnostic accuracy and treatment protocols.

The class Limnochordia harbors a single cultivated member, the mesophilic Limnochorda pilosa, which was isolated from a meromictic lake. Despite numerous molecular signatures reported in various ecosystems, the ecophysiological versatility of this deeply branched lineage of Firmicutes (Bacillota) remains poorly understood. The objective of this study was to use targeted cultivation, based on metagenome-assembled genomes from a deep terrestrial aquifer in Western Siberia, to isolate two new thermophilic members of the class. These isolates, described as Geochorda subterranea gen. nov. sp. nov. and Carboxydochorda subterranea gen. nov. sp. nov. within the Geochordaceae fam. nov., were capable of both anaerobic and aerobic respiration using fumarate and O2, respectively, with simple sugars as electron donors. The cultivated Geochordaceae have demonstrated fermentative growth and degradation of various polymers, including starch, maltose, maltodextrin, xylan, and chitin. The carboxydotrophic C. subterranea sp. nov. exhibited autotrophic growth via the Calvin–Benson–Bassham cycle, using CO, H2, and formate as electron donors and O2 as an electron acceptor, adding metabolic flexibility to the bacterium in the nutrient-depleted “deep biosphere” and supporting the possibility of aerobic metabolism in the deep subsurface. The broad physiological potential deciphered from physiological experiments and comparative genomic data explains the widespread distribution of uncultivated members of the class Limnochordia in various ecosystems, where they can oxidize complex organic substrates through both aerobic and anaerobic respiration, as well as pursue a chemolithotrophic lifestyle through the oxidation of H2 or CO.

Anaplasmataceae bacteria are emerging infectious agents transmitted by ticks. The aim of this study was to identify the molecular diversity of this bacterial family in ticks and hosts, both domestic and wild, as well as blood meal sources of free-living ticks in northeastern Paraguay. The bacteria were identified using PCR-HRM, a method optimized for this purpose, while the identification of ticks and their blood meal was performed using conventional PCR. All amplified products were subsequently sequenced. The bacteria detected in the blood hosts included Ehrlichia canis, Anaplasma platys, and Anaplasma phagocytophilum, Candidatus Anaplasma boleense, and Wolbachia spp., which had not been previously reported in the country. Free-living and parasitic ticks on dogs (Canis lupus familiaris) and wild armadillos (Dasypus novemcinctus) were collected and identified as Rhipicephalus sanguineus and Amblyomma spp. The species E. canis, A. platys, A. phagocytophilum, and Ca. A. boleense were detected in domestic dog ticks, and E. canis and A. platys were found for the first time in armadillos and free-living ticks. Blood feeding sources detected in free-living ticks were rodents, humans, armadillos and dogs. Results show a high diversity of tick-borne pathogens circulating among domestic and wild animals in the northeastern region of Paraguay.