Publications

3972

Abstract Mycoplasma haemofelis (Mhf), Candidatus Mycoplasma haemominutum (CMhm), and Candidatus Mycoplasma turicensis (CMt) are the major feline hemotropic Mycoplasma (FHM) species identified in cats worldwide. Data concerning FHM in Egypt is limited; therefore, the current study aimed to gain further insights into disease epidemiology by investigating FHM molecular prevalence, risk factors, and hemogram abnormalities in 246 Egyptian cats (shelter-housed and client-owned) during 2022–2024. Additionally, 16 S rRNA gene-based maximum-likelihood (ML) phylogenetic analysis was performed for all CMt-positive samples for the first time in Egypt. FHM was detected in 16.3% (n = 40) of cats and typed as CMhm, Mhf, and CMt in 15.4% (n = 38), 3.6% (n = 9), and 2.4% (n = 6) of cats, respectively. Single CMhm and Mhf infections were detected in 12.6% (n = 31) and 0.8% (n = 2) of cats, respectively. Dual (Mhf and CMhm) and triple (Mhf, CMhm, and CMt) infections were found in 0.4% (n = 1) and 2.4% (n = 6) of cats, respectively. CMhm was significantly associated with single infections rather than coinfections compared to other FHM species. Male sex and anemia were identified as predictors of FHM PCR positivity. FHM-infected cats had significantly lower hematocrit %, RBC counts, and hemoglobin concentrations than non-infected ones. Egyptian CMt sequences showed 97.6–100% nucleotide identity with each other. Egyptian and reference CMt strains represented nine nucleotide sequence types clustered into three well-supported clades on the ML tree without clear geographic distinction. The data generated in this study, conducted in Egypt, is crucial for enhancing our understanding of disease epidemiology and implementing effective preventive measures.

Phytoplasmas cause infections in numerous plants in agricultural ecosystems, causing significant yield and quality losses in products. In recent years, it has been known that diseases caused by phytoplasmas cause economic losses in eggplant (Solanum melongena L.) cultivation. In Turkey, research on infections caused by phytoplasmas in eggplant growing areas is quite limited. This study was carried out to detect phytoplasma infections symptomatologically and molecularly in eggplant production areas in Şanlıurfa province. Fourteen samples were collected from eggplants exhibiting symptoms such as witches’ broom, flower abnormalities (virescence, phyllody), elongation of the pedicle, arising of new shoots from flower parts, yellowing and proliferation. Phytoplasma infection was detected in 8 symptomatic samples using 16S rRNA-specific primers, P1/P7 and R16F2n/R16R2, by direct and nested PCR. Sequence information of fragments obtained as a result of molecular studies was extracted and BLAST analyses were performed. According to nucleotide sequence similarity in the 16S rRNA gene region, it was determined that the genetic group of phytoplasma causing infection in eggplant was related to ‘Candidatus Phytoplasma solani’ (CaPsol) belonging to 16SrXII-A subgroup with 98% sequence identity. To our best knowledge, this study suggests comprehensive symptomatic diagnosis of CaPsol infecting eggplants in Türkiye.

Surveys for exotic plant pests conducted during July and August of 2023 and 2024 across 20 vineyards in 12 counties throughout Minnesota, USA, revealed that less than 2% of the approximatively 3000 vines inspected (Vitis spp., hybrid grape varieties) exhibited symptoms suggestive of phytoplasma yellows disease. Observed symptoms included yellowing of leaf lamina, downward rolling of leaf margins, and necrosis of leaf margins. To investigate a potential association between these symptoms and phytoplasmas, two symptomatic plants were selected in August of 2024 from two distinct vineyards for further analysis. Petiole tissue DNA extracts were initially tested using a phytoplasma-specific real-time PCR assay (Hodgetts et al. 2009), which confirmed the presence of phytoplasma DNA in both symptomatic plants. Subsequently, the samples underwent semi-nested PCR amplification targeting the phytoplasma 16S rRNA gene, using primers P1/16S-SR followed by P1A/16S-SR (Deng and Hiruki 1991; Lee et al. 2004). The resulting amplicons were cloned and sequenced. No amplicon was produced from an asymptomatic grapevine DNA sample included as a test control. Analysis of the 16S rRNA gene sequences revealed that each plant was infected by one of two distinct phytoplasma strains, designated MN450 and MN466. Representative sequences obtained from the two samples exhibiting interoperon heterogeneity were deposited in the GenBank database under accession numbers: PQ889195 (rrnA) and PQ889196 (rrnB) for strain MN450, and PQ889197 (rrnA) and PQ889198 (rrnB), for strain MN466. Sequence analysis using the online phytoplasma classification tool iPhyClassifier (Zhao et al. 2009) showed that the 16S rRNA gene sequences from MN450 shared 1491/1495 bp (99.73%) and 1492/1495 bp (99.80%) identity (rrnA and rrnB, respectively) with that of the ‘'Ca. Phytoplasma pruni' rrnA’ reference strain (GenBank JQ044393), identifying MN450 as a ‘'Ca. Phytoplasma pruni' rrnA’-related strain and possibly representing a new subgroup within the 16Sr group III. For MN466, the 16S rRNA gene sequences shared 1471/1482 bp (99.26%) and 1469/1482 bp (99.12%) identity (rrnA and rrnB, respectively) with that of the ‘Ca. Phytoplasma asteris’ reference strain (GenBank M30790), and identifying the phytoplasma as a ‘Ca. P. asteris’-related strain belonging to subgroup 16SrI-A. To further characterize the two phytoplasma strains, the secY gene was PCR amplified from the DNA extracts as described by Lee et al. (2010). The obtained amplicons were cloned and sequenced, and representative secY gene sequences were deposited in GenBank under accession numbers PQ899621 (MN450) and PQ899622 (MN466). BLASTn searches against the NCBI core nucleotide database revealed that the secY gene sequence for MN450 exhibited 100% identity (1614/1614 bp) with ‘Ca. P. pruni’ (GenBank KM268860). Similarly, the sequence for MN466 showed 100% identity (1287/1287 bp) with ‘Ca. P. asteris’ (GenBank CP000061). In North America, grapevine yellows disease associated with strains related to 'Ca. P. asteris' and 'Ca. P. pruni' has previously been reported in various states and referred to as North American grapevine yellows (NAGY) by Davis et al. (2015). To our knowledge, this is the first report of NAGY in Minnesota, confirming that the two phytoplasma species detected are consistently associated with the disease in U.S. vineyards. While further research is needed to evaluate the potential impacts of NAGY in Minnesota, this study highlights the broader distribution of the disease across the United States.

AbstractThe severe Asiatic form of huanglongbing (HLB), caused by “Candidatus Liberibacter asiaticus” (CLas), threatens global citrus production via the citrus psyllid, Diaphorina citri. Culturing challenges of CLas necessitate reducing D. citri populations for disease management. CLas boosts the fecundity of CLas‐positive (CLas+) D. citri and fosters its own proliferation by modulating the insect host's juvenile hormone (JH), but the intricate endocrine regulatory mechanisms remain elusive. Here, it is reported that the D. citri ecdysis‐triggering hormone (DcETH) and its receptor DcETHR play pivotal roles in the reciprocal benefits between CLas and D. citri within the ovaries, influencing energy metabolism and reproductive development in host insects; miR‐210, negatively regulates DcETHR expression, contributing to this symbiotic interaction. CLas infection reduces 20‐hydroxyecdysone (20E) levels and stimulates DcETH release, elevating JH production via DcETHR, enhancing fecundity and CLas proliferation. Furthermore, circulating JH levels suppress 20E production in CLas+ ovaries. Collectively, the orchestrated functional interplay involving 20E, ETH, and JH increases energy metabolism and promotes the fecundity of CLas+ D. citri and CLas proliferation. These insights not only broaden the knowledge of how plant pathogens manipulate the reproductive behavior of insect hosts but also offer novel targets and strategies for combatting HLB and D. citri.

ABSTRACT We report the draft genomes of five Clostridium isolates from soil and agricultural by-products, four of which are proposed as Candidatus species. Members of the genus Clostridium are of significant industrial interest, and the availability of their genome sequences facilitates the understanding and exploration of their functional potential.

Marine sediments are vast, underexplored habitats and represent one of the largest carbon deposits on our planet. Microbial communities drive nutrient cycling in these sediments, but the full extent of their taxonomic and metabolic diversity remains to be explored. Here, we analysed shallow coastal and deep subseafloor sediment cores from 0.01 to nearly 600 metres below the seafloor, in the Western Pacific Region. Applying metagenomics, we identified several taxonomic clusters across all samples, which mainly aligned with depth and sediment type. Inferring functional patterns provided insights into possible ecological roles of the main microbial taxa. These included Chloroflexota, the most abundant phylum across all samples, whereby the classes Dehalococcoida and Anaerolineae dominated deep-subsurface and most shallow coastal sediments, respectively. Thermoproteota and Asgardarchaeota were the most abundant phyla among Archaea, contributing to high relative abundances of Archaea reaching over 50% in some samples. We recovered high-quality metagenome-assembled genomes for all main prokaryotic lineages and proposed names for three phyla, i.e. Tangaroaeota phyl. nov. (former RBG-13-66-14), Ryujiniota phyl. nov. (former UBA6262) and Spongiamicota phyl. nov. (former UBA8248). Metabolic capabilities across all samples ranged from aerobic respiration and photosynthesis in the shallowest sediment layers to heterotrophic carbon utilization, sulphate reduction and methanogenesis in deeper anoxic sediments. We also identified taxa with the potential to be involved in nitrogen and sulphur cycling and heterotrophic carbon utilization. In summary, this study contributes to our understanding of the taxonomic and functional diversity in benthic prokaryotic communities across marine sediments in the Western Pacific Region.

ABSTRACT The Coxiellaceae bacterial family, within the order Legionellales, is defined by a collection of poorly characterized obligate intracellular bacteria. The zoonotic pathogen and causative agent of human Q fever, Coxiella burnetii , represents the best-characterized member of this family. Coxiellaceae establish replicative niches within diverse host cells and rely on their host for survival, making them challenging to isolate and cultivate within a laboratory setting. Here, we describe a new genus within the Coxiellaceae family that has been previously shown to infect economically significant freshwater crayfish. Using culture-independent long-read metagenomics, we reconstructed the complete genome of this novel organism and demonstrate that the species previously referred to as Candidatus Coxiella cheraxi represents a novel genus within this family, herein denoted Candidatus Paracoxiella cheracis . Interestingly, we demonstrate that Candidatus P. cheracis encodes a complete, putatively functional Dot/Icm type 4 secretion system that likely mediates the intracellular success of this pathogen. In silico analysis defined a unique repertoire of Dot/Icm effector proteins and highlighted homologs of several important C. burnetii effectors, including a homolog of CpeB that was demonstrated to be a Dot/Icm substrate in C. burnetii . IMPORTANCE Using long-read sequencing technology, we have uncovered the full genome sequence of Candidatus Paracoxiella cheracis , a pathogen of economic importance in aquaculture. Analysis of this sequence has revealed new insights into this novel member of the Coxiellaceae family, demonstrating that it represents a new genus within this poorly characterized family of intracellular organisms. Importantly, the genome sequence reveals invaluable information that will support diagnostics and potentially both preventative and treatment strategies within crayfish breeding facilities. Candidatus P. cheracis also represents a new member of Dot/Icm pathogens that rely on this system to establish an intracellular niche. Candidatus P. cheracis possesses a unique cohort of putative Dot/Icm substrates that constitute a collection of new eukaryotic cell biology-manipulating effector proteins.

The occurrence of ‘Candidatus Liberibacter’ spp. and ‘Ca. Phytoplasma’ spp. associated with blotchy mottle symptoms poses challenges to huanglongbing (HLB) diagnosis using molecular techniques. The ability to detect multiple targets simultaneously and specifically is a key aspect met by quantitative PCR (qPCR). A set of primers and hydrolysis probes useful in either single or multiplex reactions for the detection and quantification of HLB-associated bacteria were developed. Sequences from conserved genes of the ribosomal proteins for Liberibacter and phytoplasma circumvent the lack of specificity and cross-reactivity problems related to 16Sr DNA gene amplification, allowing precise and specific detection of HLB-associated bacteria in citrus and in the Liberibacter vector, Diaphorina citri. The triplex reaction exhibited high quality and precision as a robust tool for quantifying ‘Ca. L. asiaticus’ (CLas), ‘Ca. L. americanus’ (CLam), and 16Sr IX phytoplasma. Triplex qPCR showed consistent results and comparable sensitivity to the ribonuclease reductase test, although quantification cycle (Cq) values were higher when compared with 16SrDNA qPCR. Detection tests using field samples indicate that the qPCR triplex can identify HLB-associated bacteria in samples with varying levels of symptoms, ranging from typical to asymptomatic. Assessment of field samples from growers indicated more than 78.6% had Cq lower than 35.0, below the cutoff established for qPCR reactions used in this work. qPCR triplex is a safe, specific, and sufficiently sensitive technique for detecting CLas, CLam, and 16Sr IX phytoplasma simultaneously, in both citrus and D. citri samples. Its application is of importance in assisting growers in making decisions for HLB management.