Publications

4485

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 X‐disease, caused by strains in the 16SrIII‐A subgroup of ‘ Candidatus Phytoplasma pruni’, is a devastating disease of Prunus species (stone fruits). Multiple outbreaks of this disease have occurred across much of North America for more than a century, with the most recent one beginning around 2010 in the Pacific Northwest of the United States, causing severe damage to the stone fruit industry. Sensitive and specific detection of X‐disease is critical to prevent the propagation and spread of infected plant material; however, current PCR‐based detection workflows lack specificity to X‐disease and require one or more PCR assays followed by additional testing to confirm the species identity. In this study, two real‐time PCR assays, one targeting the secY gene and the other targeting the tuf gene, were developed for specific detection of X‐disease in stone fruits. The CPH‐TXD assay targeting the tuf gene provided high‐sensitivity target detection of as few as five target copies with significantly reduced cross‐reactivity compared to current methods. The CPH‐SXD assay targeting the secY gene detected a minimum of 30 target copies and showed no cross‐reaction with closely related strains. The repeatability and reproducibility of both assays were verified with acceptable results. Together, these assays provide efficient and high‐specificity detection methods to combat the growing threat of X‐disease in North America.

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 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.

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.