Publications

4499

Huanglongbing (HLB), caused by Candidatus Liberibacter asiaticus (CLas), is the most destructive citrus disease worldwide, severely reducing yield and fruit quality. Although no naturally resistant cultivars are available, citrus plants overexpressing Arabidopsis NPR1 ( At NPR1) display enhanced tolerance, yet the molecular mechanisms underlying this phenotype remain insufficiently understood. To uncover early transcriptional events associated with tolerance, we performed comparative RNA-seq and phytohormone profiling of susceptible wild-type (WT) and tolerant At NPR1-overexpressing ( At NPR1-OE) Duncan grapefruit at 0, 6, and 24 hours post infection (hpi). Before infection, At NPR1 plants downregulated genes involved in cytoskeleton organization, cell wall biogenesis, and receptor signaling, suggesting a primed basal defense state. After CLas exposure, At NPR1 plants exhibited stronger and earlier transcriptional reprogramming, with substantially more differentially expressed genes at 6 hpi than WT. At 24 hpi, At NPR1 plants showed suppression of callose synthase genes and selective induction of β-1,3-glucanases, indicating more controlled phloem callose regulation. Concurrently, attenuated expression of respiratory burst oxidase homologs and ROS-associated genes suggested a moderated and less damaging oxidative burst. At NPR1 plants maintained stable levels of salicylic acid, and gibberellins while preventing the CLas-induced induction of abscisic acid observed in WT. Collectively, these findings reveal that At NPR1 overexpression enhances HLB tolerance by integrating early transcriptional reprogramming with balanced structural, oxidative, and hormonal responses. This study provides a mechanistic framework for understanding NPR1-mediated tolerance to CLas during the initial stages of infection.

Abstract Members of the phylum Deferribacterota inhabit diverse environments, but their symbiosis with protists has never been reported. We discovered an ectosymbiotic clade of Deferribacterota specifically associated with spirotrichonymphid protists in the guts of the termites Reticulitermes speratus and Hodotermopsis sjostedti and trichonymphid protists in the gut of the wood-feeding cockroach Cryptocercus punctulatus. The ectosymbiotic Deferribacterota were spiral shaped and attached to 16%–91% of the host protist cells. These formed a monophyletic cluster within an uncultured insect gut-associated family-level clade, which is sister to the vertebrate gut-associated family Mucispirillaceae. The complete genome of an ectosymbiotic Deferribacterota was obtained from a Trichonympha acuta cell in a C. punctulatus gut and analyzed together with a single-cell amplified genome of another ectosymbiotic Deferribacterota associated with Holomastigotes sp. in the gut of R. speratus. Genome analyses suggest that these Deferribacterota ferment monosaccharides and conduct fumarate and oxidative respiration with H2 as an electron donor. They thus possibly contribute to the removal of hydrogen and oxygen to protect the fermentative activity of the protist hosts. The ectosymbionts possess reduced signal transduction gene repertoires, implying that the association has provided a relatively stable environment for these bacteria. The ectosymbionts likely possess flagella with an unusually expanded number of flagellin variants up to 40, which may reflect an adaptation to their ectosymbiotic lifestyle. We propose a novel genus, Termitispirillum, for these ectosymbionts and a novel family, Termitispirillaceae, for the insect-gut clade, under SeqCode. Our findings provide new insights into the ecology and evolution of Deferribacterota.

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 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 here as Candidatus Dermatophostum, which shows specific preference for high-phosphorus environments. Its representative species, Ca. Dermatophostum ammonifactor, was enriched in the EBPR reactor and its PAO phenotype was confirmed by polyphosphate staining and fluorescence in situ hybridization. Integrative meta-omics combining 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 wastewater treatment plants worldwide, underscoring both its diverse metabolic capabilities and potential engineering implications for mitigating nitrous oxide (N2O) emissions for EBPR system. 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 Nitrobacter strain NHB1 is a nitrite-oxidizing bacterium previously demonstrated to form a consortium capable of nitrification under acidic conditions when cocultivated with a neutrophilic ammonia-oxidizing bacterium. Here, we characterize the growth of isolated NHB1 under different pH and nitrite (NO2−) concentrations, as well as its influence on the activity of obligately acidophilic soil ammonia-oxidizing archaea (AOA) isolated from acidic soils when grown in coculture. NHB1 is acidotolerant with optimal growth at pH 6.0 (range: 5.0–7.5) at an initial NO2− concentration of 500 μM. However, at lower NO2− concentrations, closer to those found in soil, its pH optimum decreases to 5.0 and with detectable growth extended to pH 3.5. In coculture, NHB1 enhances the growth of the acidophilic AOA Nitrosotalea devaniterrae Nd1 and Nitrosotalea sinensis Nd2, which are highly sensitive to NO2-derived compounds and typically oxidize only ~200 to 300 μM ammonia (NH3) when grown in batch cultures as isolates. However, in coculture with NHB1, both strains oxidized up to ~3 mM NH3, limited only by the buffering capacity of the medium, and their pH range was also extended downward by ~0.5 units. NHB1 also possesses a cyanase, enabling reciprocal cross-feeding through cyanate-derived NH3 production while utilizing AOA-derived NO2−. These findings suggest that NO2− removal is essential for ammonia oxidizer growth in acidic soils and emphasize the importance of considering substrate and metabolic product concentrations when characterizing ecophysiology. Genome analysis reveals that NHB1 is distinct from validated species, and we propose the name “Nitrobacter laanbroekii.”

An anaerobic, salt-tolerant, rod-shaped bacterium, strain JL216-2 T , was isolated from the shell of the hydrothermal gastropod Alviniconcha marisindica in the Tianxiu hydrothermal field on the Carlsberg Ridge, Indian Ocean. Cells of strain JL216-2 T were motile and 0.5×3–6 µm in size. Strain JL216-2 T grew at 10–32 °C (optimal 28 °C), pH 6.0–9.0 (optimal pH 7.0) and 0.5–12.0% (w/v) sodium chloride(NaCl, optimal 2.5%). Strain JL216-2 T exhibited metabolic versatility, being capable of heterotrophic growth on complex organic compounds. It also performed autotrophic growth by utilizing carbon dioxide (CO₂) fixation or sodium bicarbonate (NaHCO 3 ) as the sole carbon source, driven by hydrogen oxidation coupled with Fe(III) reduction. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the strain was most closely related to Helicovermis profundi S502 T (90.43% sequence similarity). The genome size of strain JL216-2 T was 4.91 Mb, with a G+C content of 39.54 mol%. Phylogenomic analysis and overall genome relatedness indices values indicated a close relatedness of strain JL216-2 T to H. profundi S502 T and Fusibacter spp. The combined genotypic and phenotypic data showed that strain JL216-2 T represents a novel genus within the order Eubacteriales . The type strain is JL216-2 T (=MCCC M30262 T =KACC 23553 T ).

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 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.