Publications

4560

Abstract Background ‘ Candidatus Liberibacter solanacearum’ (Lso) is a phloem-limited bacterial pathogen causing significant diseases in solanaceous crops. In the United States, haplotypes A and B are transmitted by the potato psyllid Bactericera cockerelli . We previously identified differences in their acquisition and transmission between adults and nymphs. The present study characterized the dynamics of LsoA and LsoB acquisition and transmission by nymphs and examined the transcriptional responses of the nymphal gut upon their acquisition. Methods and results Nymphs were exposed to LsoA- or LsoB-infected plants for 1, 3, 5, or 7 days to measure the bacterial accumulation and for 8 days to assess the transmission efficiency following sequential inoculation of tomato plants. Quantitative PCR showed that LsoB accumulated to higher levels than LsoA after 3 days of acquisition. Following the sequential inoculation, LsoB was transmitted earlier than LsoA indicating a shorter latency period. RNA-seq analysis of the guts following a 1- and 5-day acquisition access periods revealed a greater transcriptional regulation at 5 days than at 1 day. Furthermore, the responses were haplotype-specific: LsoA primarily affected genes involved in protein translation, ER stress, and cell cycle regulation, whereas LsoB regulated genes involved in autophagy, apoptosis, and immune pathways. Conclusions This study revealed haplotype-specific gene regulation potentially leading to LsoB being transmitted more efficiently by psyllid nymphs.

Abstract Bovine hemoplasmas, including Mycoplasma wenyonii (M. wenyonii) and Candidatus Mycoplasma haemobos (C. M. haemobos) , are vector-borne pathogens that can cause significant clinical diseases in cattle. This case report from Türkiye describes the clinical findings, hematological analysis, and serum biochemistry results of a cow presenting with hemoglobinuria caused by C. M. haemobos . Blood samples from the infected animal were analyzed using hematological, biochemical, microscopic, and molecular methods. Multiplex polymerase chain reaction assay confirmed the infection, and microscopic analysis revealed a parasitemia rate of 6.99%. Clinical signs included hemoglobinuria, icterus, and anemia. The treatment included fluid therapy, antibiotic (oxytetracycline), and antianemic drugs. The cattle died 1 month post-treatment, reportedly due to a lower respiratory tract infection. This case report describes the diagnostic methods, clinical findings, and laboratory results. A widescale molecular survey of C. M. haemobos in cattle is warranted, with investigation of its pathogenicity and pathogenies.

Abstract The pear psyllid Cacopsylla pyri is the main vector of ‘ Candidatus Phytoplasma pyri’, the causal agent of pear decline, a disease associated with severe yield losses in pear production. Phytoplasmas are further known to interfere with their vectors metabolism; therefore, we investigated whether an infection with ‘ Ca. P. pyri’ is associated with changes in nutrient storage of the pear psyllids. Additionally, we measured whether the phytoplasma infection is associated with differences in flight performance in C. pyri , as the flight activity of the vector is a key component of phytopathogen spread. Using a flight mill, we quantified flight distance, duration, and speed, and combined qPCR-based infection detection with colorimetric assays to determine protein, carbohydrate, and lipid content from the same individual. Infected psyllids contained significantly higher levels of soluble carbohydrates than uninfected individuals. Lipid content was not affected by the phytoplasma infections but was found to be consistently higher in males than in females, while protein levels did not vary with sex or infection status. Despite infection-related differences in carbohydrate levels, phytoplasma infection did not significantly affect flight duration, distance, or speed. These results indicate that infection-associated shifts in bulk carbohydrate storage do not translate into altered flight motivation or performance, suggesting a functional decoupling between metabolic state and realised dispersal capacity under standardised laboratory conditions.

Citrus huanglongbing (HLB) is recognized as the most destructive bacterial disease affecting citrus. Among the causal agents, Candidatus Liberibacter asiaticus (Las) is the predominant species causing HLB epidemics worldwide. Detecting this phloem limited bacterium at early or low titer stages and assessing its cellular activity remain major challenges for HLB research and management. To address these limitations, we developed a one step reverse transcription quantitative PCR (RT qPCR) assay for highly sensitive Las detection using total nucleic acids and the established Li 16S rRNA/rDNA primer–probe set. This method simultaneously amplifies both Las 16S rRNA and rDNA in a single reaction, increasing sensitivity by up to 500 fold compared with qPCR, which targets only 16S rDNA. The improved sensitivity reduced false negatives, expanded sampling capacity, and enabled detection of low titer infections previously undetectable. In addition, the higher abundance of 16S rRNA generated a consistent Ct gap between RT qPCR and qPCR, which we used to estimate relative Las cell activity in vivo in host insects and plants and to identify the most effective inoculum sources. This comparative detection strategy was further applied to evaluate inoculum potential, monitor changes in relative cell activity during in vitro cultures, and assess antimicrobial treatments targeting Las. The persistent detection of long lasting, low titer Las infections underscores the complex biology of the HLB causal agent and highlights ongoing challenges for effective disease management.

Abstract Environmental genomics has led to the discovery of many new lineages of archaea, including “DPANN” (or Nanobdellati), comprising organisms with small genomes, reduced gene content, and potentially symbiotic or parasitic lifestyles. DPANN live in various environments, and several lineages have been identified that are adapted to extremely high salt concentrations, including the Nanohaloarchaeota. Since it was long thought that the Haloarchaea (within ‘Euryarchaeota’) were the only high salt-adapted archaea, the origins of these genome-reduced halophiles have been debated. Here we used phylogenetic, comparative genomic, and gene-tree/species-tree reconciliation approaches to resolve the evolution of halophily within DPANN, making use of recently-published genomes that help to inform the phylogenetic placement and genome evolution of salt-adapted lineages. Phylogenetic analysis placed Nanohaloarchaeota sister to a previously uncharacterised lineage, which we here refer to as Terrarchaeota. Terrarchaeota appear to be predominantly anaerobic thermophiles that are not adapted to high salt concentrations, indicating that adaptation to high salt evolved after their divergence from Nanohaloarchaeota. Furthermore, our analyses identified genomic hallmarks of salt adaptation in another recently discovered halophilic DPANN lineage within Aenigmatarchaeota, the Haloaenigmatarchaeaceae. We found that the Nanohaloarchaeota and Haloaenigmatarchaeaceae have distinct sets of proteins that enable life at high salt concentrations but share a common mechanism of evolutionary adaptation, in which niche-relevant genes were acquired horizontally from their halophilic hosts. This work provides the first detailed investigation into the enigmatic Terrarchaeota, and new insights into the convergent evolution of high salt adaptation within symbiotic clades of Archaea.

Abstract Mauritius is one of the two countries where two huanglongbing (HLB)-associated bacteria, ‘ Candidatus Liberibacter asiaticus’ (CLas) and ‘ Ca . Liberibacter africanus’ (CLaf), have coexisted since at least the 1990s, offering insights into their interactions, particularly given their divergent thermal tolerances. The objective of the study was to revisit the unique HLB epidemiological situation in Mauritius using modern diagnostic and genotyping techniques in the broader context of global warming. Using multiple-locus variable number tandem repeat analysis (MLVA), multilocus sequence analysis (MLSA), a real-time PCR assay, and a loop-mediated isothermal amplification (LAMP) assay, we detected both species. While CLas was commonly detected from sea level up to 580 m above sea level (masl), CLaf was commonly detected between 300 and 600 masl, where temperatures appear higher than in African regions where CLaf has been reported, suggesting putative adaptation to warmer temperatures of Mauritian CLaf. LAMP data also identified some limitations in inclusivity in Keremane’s system. Based on tandem-repeat (TR) data, a single population of CLas was identified whose level of diversity did not differ statistically from that of Réunion, a geographically proximate island with a similar epidemiological situation. These two populations were only weakly differentiated, consistent with the detection of very few private alleles in Mauritius. Prophage- and miniature inverted-repeat transposable element (MITE)-based genotyping confirmed the high genetic relatedness between samples from these islands. Altogether, our results suggest a putative identical source for CLas and/or epidemiological links between these islands. We discuss the implications of our results for surveillance and management of HLB.

Abstract Members of the bacterial family Endozoicomonadaceae are ubiquitous marine symbionts associated with diverse hosts, including corals, sponges, molluscs, and fishes. Despite their ecological pervasiveness, inhabiting deep-sea vents and sunlit coral reefs, their taxonomy remains inconsistent, largely due to reliance on 16S rRNA gene phylogenies that fail to fully resolve evolutionary relationships. Here, we integrate phylogenomics, comparative genomics, and overall genome relatedness indices across 63 high-quality genomes to re-define genus boundaries within the Endozoicomonadaceae family. Among the tested metrics, the core-proteome average amino acid identity proved the most robust for genus-level resolution, with a proposed identity cutoff of 76%, aligning with phylogenomic structure and maintaining taxonomic robustness across the family. Phylogenomic reconstructions further revealed that the taxon Endozoicomonas is paraphyletic, forming two well-supported, but distinct clades. This prompts a taxonomic revision, dividing Endozoicomonas into Endozoicomonas sensu stricto and Neoendozoicomonas gen. Nov., with Neoendozoicomonas montiporae designated as the type species. Furthermore, the sister clade to Endozoicomonas sensu stricto - comprising the genera Endonucleibacter and Sororendozoicomonas - is characterized by genomic streamlining, including reduced genome size, lower GC content, reduced number of orthogroups, and potential functional divergences consistent with host specialization. Functional annotations highlighted secretion and conjugation systems as key differentiators between genera, emphasizing potential distinct host-interaction strategies. This genome-based framework refines the taxonomy of Endozoicomonadaceae, provides additional criteria for genus delimitation, and strengthens the evolutionary and ecological interpretation of these widespread marine bacterial symbionts.

ABSTRACT Acidophilic ammonia-oxidizing bacteria (AOB) have only recently been discovered. These organisms hold great promise for acidic wastewater treatment; however, their physiology remains poorly understood compared to that of neutrophilic AOB. Here, we investigated the physiology of the acidophilic AOB “Candidatus Nitrosacidococcus tergens ” sp. RJ19 across a broad pH range (2.5–7.0) using a specialized bioreactor system. We monitored nitrogen (N) transformations combined with microbial community composition and transcriptomic profiles, focusing on nitrogen metabolism and proton stress responses. Our results show that above pH 6.0, “ Ca . Na. tergens ” performs complete and stoichiometric conversion of ammonium to nitrite, coinciding with isotopic fractionation effects specific for ammonia oxidation and increased expression of key ammonia oxidation genes. The apparent absence of nirK and cycA did not impede ammonia oxidation, suggesting that these genes are non-essential in this context. Below pH 6.0, nitric oxide and nitrate accumulated, and nitrous oxide (N₂O) levels, although negligible compared to the other N-compounds, peaked near pH 4.0. Stable isotope analysis, including the site-specific 15 N-enrichment at the inner (α) and outer (β) nitrogen positions of the N 2 O molecule, indicated nitrifier-denitrification as the source of N₂O, supported by the highest norB expression at this pH. These findings provide new insights into the acid-tolerant physiology of “ Ca . Na. tergens ” and advance its potential application in engineered nitrogen removal systems under acidic conditions. IMPORTANCE The world is facing a climate crisis intensified by human-driven nutrient pollution. Ammonia and the bacteria that oxidize it are central both to the global nitrogen cycle and to wastewater treatment. The acidophilic ammonia oxidizer “ Candidatus Nitrosacidococcus tergens” was previously shown to oxidize ammonia under highly acidic conditions; however, a complete understanding of its metabolism is lacking. Our study now shows that “ Ca . Na. tergens” performs canonical ammonia oxidation across a broad pH range. At pH values below 6, however, a combination of chemical and biological processes leads to the production of nitrate, nitric oxide, and the greenhouse gas nitrous oxide. In addition, we show that these bacteria adapt to proton stress through mechanisms beyond transcriptional mechanisms. Our study highlights the robust metabolism of acidophilic ammonia oxidizers and expands our understanding of nitrification under acidic conditions.

Abstract ‘ Candidatus Liberibacter solanacearum’ (Lso) is a devastating bacterial pathogen of crops transmitted by psyllids. Previously, a link between changes in ubiquitination and the progression of diseases caused by Lso was established. Among the Lso haplotypes, LsoA and LsoB infect solanaceous crops. LsoA induces chlorosis and stunting in tomato, while LsoB infection leads to more severe symptoms, including plant death. To better characterize the molecular basis of the differences in the diseases caused by these haplotypes, we cataloged the ubiquitinome of tomato leaves four weeks after LsoA and LsoB-infection and following infestation with Lso-free psyllids. Our analysis revealed haplotype-specific changes in the plant ubiquitinome. Gene Ontology (GO) terms related to glucose metabolism and proteasome-mediated ubiquitin-dependent protein catabolism were enriched among the differentially ubiquitinated proteins between LsoA-infected and Lso-free infested plants. Additionally, a KEGG pathway analysis determined an overrepresentation of proteins involved in carbon metabolism and fixation via the Calvin cycle. In contrast, the comparison of LsoB-infected and Lso-free infested plants revealed a broader range of enriched GO terms and KEGG pathways, including those associated with carbohydrate and energy metabolism, proteasome activity, and oxidative stress. These results contribute to our understanding of the development of Liberibacter-related diseases and reveal the importance of ubiquitin-related pathways in Lso infection. They also contribute to the expanding inventory of ubiquitinated proteins.