Publications

4481

Abstract BACKGROUND Huanglongbing (HLB) is the most devastating citrus disease worldwide, with no resistant commercial citrus varieties identified and no effective chemical treatments currently available on the market. The main challenge in developing effective strategies for controlling HLB is the inability to culture its associated pathogen, the fastidious Candidatus Liberibacter asiaticus ( C Las), in defined media or axenic cultures. RESULTS In this study, we developed an effective method for screening antimicrobials against C Las using periwinkle as a model plant. Compared with citrus, periwinkle offers notable advantages, including reduced time, increased efficiency, and lower costs. This method consists of two main components: screening exogenously applied antimicrobials and evaluating overexpressed endogenous antimicrobial peptides. Using this method, we demonstrated that bromothalonil and cyproconazole effectively inhibit the proliferation of C Las in hydroponically grown periwinkle. Further analyses revealed that bromothalonil and cyproconazole were also highly toxic to other citrus pathogens, including Xanthomonas citri subsp. citri ( Xcc ), Alternaria alternata tangerine pathotype (Z7), and Penicillium digitatum (KH8). In addition, we established an Agrobacterium rhizogenes ‐mediated hairy root genetic transformation system for periwinkle and demonstrated that the proliferation of C Las was significantly inhibited in transgenic roots overexpressing antimicrobial peptide (SAMP). CONCLUSION An efficient antimicrobial screening system for C Las and several other citrus pathogens has been established. By addressing challenges such as high costs, prolonged experimental periods, and heavy workloads, we anticipate that this developed method will serve as a useful tool for the identification of antimicrobials effective against C Las and other citrus pathogens. © 2025 Society of Chemical Industry.

Background: Ticks are significant vectors of pathogens, and their nonpathogenic intracellular bacteria, known as endosymbionts, influence tick physiology and may interact with tick-borne pathogens. Such relationships can affect transmission dynamics and understanding them is essential for designing strategies that control tick-borne diseases and reduce their impact on human and animal health. Aim: This study aimed to determine the prevalence and density of two key endosymbiotic bacteria, Francisella-like endosymbiont (FLE) and Candidatus Midichloria mitochondrii (Ca. M. mitochondrii), and to investigate possible correlations between these symbionts and the protozoan parasite Theileria annulata in Hyalomma anatolicum ticks. Methods: Between January and October 2024, 116 ticks were collected from cattle in the Diwaniyah province, Iraq. The morphological examination was followed by molecular confirmation using mitochondrial 16S rRNA sequencing. Quantitative polymerase chain reaction was applied to detect and quantify FLE, Ca. M. mitochondrii, and T. annulata in H. anatolicum. Results: H. anatolicum was the predominant species, representing 86% of ticks, whereas H. excavatum accounted for 13%. Mitochondrial 16S rRNA sequences confirmed species identity and showed 99.48-100% similarity to reference isolates from China, Pakistan, Turkey, India, and Russia. FLE was detected in 100% of H. anatolicum, with mean loads of 7.9× 103 copies per tick. Ca. M. mitochondrii was detected in 99% of specimens, with mean loads of 3.7×102 copies per tick. FLE densities tended to be higher in females compared to males (9.7 × 10³ copies vs. 1.8 × 10³ copies) but difference was not statistically significant. Ca. M. mitochondrii loads also differed significantly (4.8 × 10² copies vs. 1.3 × 10¹ copies). T. annulata was present in all ticks, with the highest copy numbers in semi-engorged females (1.9 × 104 copies), followed by males (2.8 × 10³ copies). Our findings revealed a statistically significant association (p<0.05) between T. annulata and Ca. M. mitochondrii, whereas no significant correlation was observed between T. annulata and FLE. Conclusion: The presence of FLE and Ca. M. mitochondrii in H. anatolicum from the Diwaniyah province was documented. Evidence indicated associations between symbiont density and T. annulata infection, and the results point toward a possible role of endosymbiotic bacteria in pathogen persistence and the epidemiology of tick-borne diseases.

Abstract Dermatophilaceae polyphosphate-accumulating organisms (PAOs), formerly classified as Tetrasphaera PAOs, play pivotal roles in enhanced biological phosphorus removal (EBPR). However, their phylogenetic diversity, ecological preferences, and metabolic traits remain poorly characterized, and a robust marker gene for their classification is lacking. Here, we performed an extensive phylogenomic and metabolic analysis of Dermatophilaceae PAOs utilizing 46 newly recovered metagenome-assembled genomes (MAGs) from a laboratory-scale EBPR reactor treating high-strength wastewater and full-scale wastewater treatment plants. These analyses revealed a previously uncharacterized PAO genus, named Candidatus Dermatophostum, which is adapted to high-phosphorus environments. Its representative species, Ca. D. ammonifactor, was enriched in the EBPR reactor and its PAO phenotype was confirmed by polyphosphate staining and fluorescence in situ hybridization. Genomic, transcriptomic, and protein structure analyses revealed its specialized metabolic capabilities for phosphate metabolism, glycogen synthesis and dissimilatory nitrate reduction to ammonium. Moreover, Ca. Dermatophostum was found to be widely distributed across WWPTs worldwide, underscoring both its ecological importance and its potential role in mitigating nitrous oxide (N 2 O) emissions. Finally, we propose a ppk1 -based classification framework that resolves Dermatophilaceae PAOs into six distinct clades, consistent with whole-genome phylogeny, and demonstrates that ppk1 can serve as a reliable marker gene for tracking these populations. Together, these findings expand the ecological and functional understanding of Dermatophilaceae PAOs and highlight their promise for advancing sustainable wastewater treatment and resource recovery.

ABSTRACT Recent discoveries of aerobic methanotrophs in non-seep carbonate-rich environments in the deep sea suggest that these organisms may persist as part of the rare biosphere. Recovering rare, active methanotrophs through targeted culturing is essential for understanding their persistence under the oligotrophic non-seep conditions and for uncovering their genomic adaptations related to the survival in energy-limited ecosystems. In our study, using metagenomic analysis of enrichment cultures from the Alpha Crucis Carbonate Ridge, we discovered Methylotuvimicrobium crucis sp. nov., a novel methanotroph representing the rare biosphere in native sediments, described in accordance with the SeqCode rules. Recent discoveries of aerobic methanotrophs in non-seep carbonate-rich environments in the deep sea suggest that these organisms may persist as part of the rare biosphere. Recovering rare, active methanotrophs through targeted culturing is essential for understanding their persistence under the oligotrophic non-seep conditions, and for uncovering their genomic adaptations related to the survival in energy-limited ecosystems. In our study, using metagenomic analysis of enrichment cultures from the Alpha Crucis Carbonate Ridge, we discovered Methylotuvimicrobium crucis sp. nov., a novel methanotroph representing the rare biosphere in native sediments, described in accordance with the SeqCode rules. Phylogenomic analysis revealed <95% of Average Nucleotide Identity (ANI) to described species, with genomic evidence of deep-sea specialization including: (i) stress adaptation through cold-shock proteins (CspA) and DNA repair systems (UvrD/LexA), (ii) metabolic versatility via complete methane oxidation (pmoABC), nitrogen fixation (nifHDK), and sulfur cycling (sox/sqr) pathways, and (iii) niche partitioning through biofilm formation (GGDEF/EAL) and heavy metal resistance (CopZ/CzcD). Comparative genomics identified a 1,234-gene deep-sea core shared with Methylotuvimicrobium sp. wino1, enriched in mobile elements (TnpA, prophages) suggesting horizontal gene transfer drives adaptation. While undetected in situ amplicon surveys, Methylotuvimicrobium crucis exhibited enrichment under methane availability, demonstrating its role as a latent methane filter. These findings contribute to the understanding of the ecological significance of aerobic methanotrophs in deep-sea systems, revealing how rare microbial taxa with genomic plasticity have the potential to influence biogeochemical cycling in deep carbonate-rich environments. IMPORTANCE Microbial communities in deep-sea sediments play crucial roles in global biogeochemical cycles, yet they remain poorly characterized due to the challenges of sampling and culturing under extreme conditions. This study provides a comprehensive overview of microbial diversity and functional potential in carbonate-rich deep-sea sediments, with an emphasis on methane-oxidizing bacteria. By combining high-throughput metagenomics and comparative genomics, we reconstructed high-quality genomes from previously uncharacterized microbial consortia, including novel members of the genus Methylotuvimicrobium . Our findings shed light on the ecological strategies of methanotrophs in oxygen-limited environments and expand the genomic representation of key players in carbon cycling.

Abstract Deciphering the genomic basis of ecological diversification in activated sludge microbiomes is essential for optimizing treatment technology and advancing microbial ecology. Here, we present a global genome-resolved investigation of Candidatus Accumulibacter, the primary functional agent of enhanced biological phosphorus removal, based on 828 metagenomes from wastewater treatment plants across six continents. We recovered 104 high-quality Candidatus Accumulibacter metagenome-assembled genomes, discovering a new clade (Clade IV), substantially expanding the known phylogenetic diversity and revealing a ubiquitous yet geographically heterogeneous global distribution. Phylogenomic and pangenome analyses uncovered extensive clade-specific gene gain and loss, particularly in nitrogen metabolism, suggesting divergent evolutionary trajectories shaped by relaxed selection and niche adaptation. Genome-wide patterns of convergent streamlining and enriched antiviral defense systems indicate selective pressures from strong competition and viral predation. Constraint-based metabolic modeling revealed pervasive amino acid autotrophies and metabolic complementarity, coupled with distinct carbon utilization strategies that support ecological specialization across operational settings. Experimental validation reconciled model-phenotype discrepancies, highlighting the importance of transporter promiscuity and gene regulation in carbon substrate assimilation. Collectively, our findings redefine Candidatus Accumulibacter as a dynamic model of microbial genome plasticity, metabolic adaptation, and ecological resilience, providing an insight for understanding how microbial communities adapt and respond under engineered environmental conditions.

Background/Objective. This study analyzed for the first time the asymptomatic condition induced by CLas in Citrus sinensis / C. aurantium based on initial reports in the Central Gulf region of Mexico. The objective was to analyze with a mechanistic approach the spatial-temporal concentration of CLas at the plant level in commercial orchards to demonstrate the possible implication of a prolonged incubation period in the asymptomatic expression of HLB. Materials and Methods. Two out of three trees (A1-A3) (>30 years old) of asymptomatic Valencia sweet orange trees positive for CLas, identified through official diagnostic protocols in San José Acateno, Puebla, were confined. On trees A2 and A3, 13 and 16 secondary branches were marked, with a total of 112 and 108 foliage/samples per month, respectively (total 646 samples), obtained from the distal and proximal parts of each branch. Seven monthly samplings of plant material were carried out between May and December. At the root level, three categories were sampled by diameter/function and by a combination of six distal trunk sectors (0 - 280 cm) by 4 in depth (0 x 110 cm) with a total of 111 samples. DNA extraction was performed with 2% CTAB and quantification by RT-qPCR (oligos, HLBp/HLBas). Results. In canopy, the incidence/branches in A3 was 52.2% (Ct = 22.7 – 34.9), and in A2 47.8% (Ct = 23.4 –35), with no statistical difference (p = 0.48). The inoculum load was 1.6 (±1.3) log-CLas, equivalent to 109 copies/reaction in 100 mL (range in number of copies 6.3 to 68,870.7). CLas did not differ between distal (1.6 log) and proximal (1.5 log) samples (p = 0.33). Significative temporal fluctuation was found between June and October in positive samples (37 – 87, p < 0.0001), but not in CLas concentration (1.6 – 3.5 log-CLas, p > 0.72). The distribution of CLas by branch had a temporal effect (p = 0.0001) and by sector (p = 0.07), showing high mobility and seasonality. The maximum concentration (Ymax) was associated with the middle-upper sector of the canopy, and high mobility towards the lower and upper sectors in Yinitial and Yfinal. In roots, 38.7% positivity was found. The detectability of CLas was homogeneous among root types (p < 0.0001) but variable in concentration (p = 0.04). The 47.6%, 15.4%, and 38.7% incidence was associated with 38.3, 43.4, and 9.5 average CLas copies for support, conduction, and absorption roots, respectively (p = 0.0001). Two sections with higher CLas intensity-concentration (80 – 144 copies) were associated with roots in the middle drip zone (160 – 240 cm) and medium depth strata (50 – 80 cm). Conclusion. CLas was significantly and consistently detected in plant and root tissue throughout the experimental period, but at fluctuating, non-incremental concentration, analogous between trees A2 and A3. The wide canopy-root distribution suggests a chronic systemic effect. Consequently, the incubation period does not determine the asymptomatic condition in the sweet orange/sour orange productive system investigated. Other factors, e.g. inherent to pathogen variability, could be involved. These results contribute to optimizing a surveillance-monitoring system considering seasonality, zone-branch, sector-canopy and type of tissue, with a greater probability of detection of CLas even in an eventual asymptomatic condition and generate new insights for Clas management.