Publications

4855

Abstract Background The specific pathophysiological drivers and host–pathogen–environment factors that dictate the pathogenicity of Candidatus Mycoplasma haemominutum (CMhm) remain poorly defined, hindering accurate clinical interpretation and effective disease control. This study provides detailed mechanistic insights into CMhm-associated anaemia in naturally infected cats by integrating analyses of concurrent diseases and comprehensive clinicopathological panels, alongside molecular characterisation in an under-represented region. Two groups of CMhm-infected cats (anaemic, n = 13; non-anaemic, n = 25) were systematically screened for 14 concurrent infectious, metabolic, or endocrine conditions, alongside extensive haematological, biochemical, and iron profile assessments. Multivariable logistic regression identified independent risk factors, while CMhm molecular diversity was assessed using 16S rRNA gene sequencing. Results Multivariable analysis identified flea infestation as the sole significant driver of anaemia (OR 7.77; P = 0.04), underscoring a key vector-trigger role and contributing evidence to the ongoing debate on CMhm’s flea-borne transmission. Although specific conditions such as feline immunodeficiency virus, feline leukaemia virus, Mycoplasma haemofelis, Candidatus Mycoplasma turicensis, and hyperglycaemia were not statistically significant, they may still be biologically relevant. Notably, most cats in the non-anaemic group also had comorbidities, suggesting that disease stage or severity may be more critical than mere presence. Supporting this, the anaemic group showed significantly lower lymphocyte counts and a downward trend in other leucocytes. Anaemia appeared deceptively normocytic-normochromic and non-regenerative despite ongoing CMhm-induced haemolysis, indicating additional counteracting mechanisms. Detailed clinicopathological and concurrent disease investigations pointed to true iron deficiency (flea-related) and functional iron deficiency (inflammatory comorbidities) as contributors. The coexistence of these three opposing pathophysiological processes shaped the paradoxical haematological and iron profile. Molecular analysis identified three CMhm nucleotide sequence types (nSTs) new to the region. Conclusions The results of this study suggest that CMhm-associated anaemia is likely a secondary condition, in which environmental triggers and host immune status shape disease manifestation. This study elucidates the complex pathophysiological mechanisms underlying anaemia, providing insights to enhance clinical diagnosis and inform control strategies.

Abstract Many Patescibacteria lineages are abundant in wastewater treatment plants, yet their interaction partners and ecological roles remain poorly understood. Here, we identify a specific association between a novel Saccharimonadia genus, Candidatus Lutisaccharimonas, and the widespread polyphosphate-accumulating organism (PAO) Candidatus Phosphoribacter. Fluorescence in situ hybridization revealed close physical proximity between the two taxa, supporting an epibiotic relationship. Correlated population dynamics, CRISPR spacer matches and evidence of horizontal gene transfer further corroborated this association. Comparative genomic analyses showed that Ca. Lutisaccharimonas has greater metabolic versatility than is typical of Patescibacteria, including complete pathways for de novo nucleotide biosynthesis and genes involved in amino acid metabolism. These metabolic features may shape its interactions with Ca. Phosphoribacter. Together, our findings identify a previously undescribed host association for Patescibacteria, expand current understanding of their metabolic potential, and suggest that the interactions between Saccharimonadia and PAOs may influence phosphorus removal in engineered ecosystems.

Abstract Antimicrobial peptides (AMPs) are widely used in disease-resistant breeding, and their application in citrus has recently been reported. However, the molecular mechanism by which AMPs inhibit the Huanglongbing (HLB) remains unclear. This study aimed to clarify the inhibitory mechanism of Hinnavin I (an AMP) against HLB-associated pathogens. We first demonstrated that Hinnavin I exhibits antibacterial activity against several tested Gram-negative bacterial strains. Using a transgenic citrus hairy root system, the heterologous expression of Hinnavin I was found to significantly reduce Candidatus Liberibacter asiaticus (CLas) colonization. Transcriptomic and phytohormone analyses further showed that constitutive Hinnavin I expression was associated with altered defense-related gene expression and enhanced SA-related responses in citrus hairy roots. Multiple interaction assays identified the CLas outer membrane lipoprotein Omlp as a direct interaction partner of Hinnavin I both in vitro and in vivo.

Introduction Citrus huanglongbing (HLB), caused by Candidatus Liberibacter asiaticus (CLas), is a globally devastating plant disease characterized by severe disruption of host physiology. Although alterations in plant hormone homeostasis, including salicylic acid and abscisic acid (ABA), are hallmarks of HLB, the contribution of plant-associated microbiota to these hormonal changes remains poorly understood. Here, we investigated whether ABA depletion in HLB-affected citrus is associated with microbial metabolism. Methods ABA and its metabolites were quantified in HLB-affected citrus tissues by liquid chromatography–mass spectrometry. The capacity of CLas strain Ishi-1 and Flavobacterium spp. strains CGRL1 and CGRL2 to metabolize ABA was assessed using in vitro culture assays. The effects of exogenous ABA on bacterial growth were also evaluated. Results ABA concentrations were significantly decreased in symptomatic HLB-affected tissues, concomitant with the accumulation of the ABA catabolite dehydrovomifoliol. Furthermore, CLas strain Ishi-1 and Flavobacterium strains CGRL1 and CGRL2 degraded ABA and produced dehydrovomifoliol, whereas no such activity was detected in non-inoculated controls. Notably, exogenous ABA promoted bacterial growth, suggesting that ABA may serve as a metabolic substrate or growth-enhancing factor for these bacteria. Discussion These findings identify microbial ABA metabolism as a potential driver of hormone depletion in HLB-affected citrus. Our results further suggest that CLas and its associated bacteria may actively reshape host hormone homeostasis through ABA metabolism, providing a mechanistic link between pathogen-associated microbial communities and hormonal imbalance during HLB development.

Phytoplasmosis associated with the ‘Candidatus Phytoplasma solani’ subgroups 16SrXII-A and 16SrXII-P (Mollicutes) poses a threat to European sugar beet cultivation, alongside the “basses richesses” syndrome caused by ‘Ca. Arsenophonus phytopathogenicus’ (Gammaproteobacteria). Both subgroups co-occur in sugar beet and have been detected in Pentastiridius leporinus, a polyphagous planthopper associated with SBR-affected fields. Despite ongoing monitoring of disease spread and pathogen characterization, approaches for direct subgroup differentiation remain limited. Consequently, discriminative conventional PCR assays targeting secY were developed, with detection limits estimated using synthetic gene fragments. Assay performance was assessed using 900 DNA templates from sugar beet from Germany and neighboring countries, 50 German red beet samples, and 320 planthoppers. Distinct amplicon sizes of 549 bp and 857 bp enabled reliable detection and differentiation of 16SrXII-A and 16SrXII-P, respectively. Detection rates were broadly concordant with those obtained using an established phytoplasma detection approach. Estimated detection limits were 100 target copies for 16SrXII-A and 1000 for 16SrXII-P. Assay application indicated more frequent detection of 16SrXII-P in samples from Germany and of 16SrXII-A in those from neighboring countries. The assays provide a rapid and accessible tool for subgroup differentiation and may facilitate large-scale epidemiological surveillance and future studies of host tolerance.

The distribution of ‘Candidatus Phytoplasma prunorum’, a phloem-limited pathogen, varies among plant tissues during the year, but knowledge about quantitative comparisons of its presence in roots and above-ground tissues across Prunus species remains limited. The variation in ‘Ca. P. prunorum’ in four Prunus species was monitored monthly during 2025 using quantitative PCR-based absolute quantification. Roots, one-year-old shoots and annual shoots were sampled monthly from 21 infected trees of Prunus armeniaca, Prunus domestica, Prunus persica and Prunus salicina. Phytoplasma titer varied with plant part and sampling month, and differences were also observed among the examined host groups. Because cultivar and rootstock were linked to species, these differences cannot be attributed to species alone. Roots provided the most consistent year-round detection, whereas above-ground tissues showed stronger monthly variation during the studied period. P. domestica displayed the most distinct pattern: roots remained frequently positive, while one-year-old shoots and particularly annual shoots had lower detection rates and lower phytoplasma titers. Multilocus genotyping detected ten multilocus profiles, but their uneven distribution among only 21 trees precluded a reliable evaluation of their relationship with phytoplasma titer or symptom expression.

Rapid and accurate diagnosis of ‘ Candidatus Phytoplasma’ is critical for disease management but remains challenging due to the pathogen's unculturable nature, low titers, uneven distribution, and genetic complexity. Although current official guidelines provide a highly adaptable taxonomic foundation for species assignment, translating these broad recommendations into standardized routine diagnostics remains difficult. This study implemented the established species assignment guidelines by developing a structured, proof-of-concept diagnostic framework utilizing a multilocus sequence analysis workflow coupled with Oxford Nanopore Technologies (ONT) sequencing. The developed workflow targets four barcodes—16S, secA, rplV, and tufB—and replaces fixed demarcation criteria with a best-match principle for non-16S barcodes. Furthermore, the study offers guidance for barcode selection and challenges the use of 16S rRNA sequencing as a sole screening assay. Moreover, a read-based quantification system incorporating control thresholds was implemented to provide an objective and reliable assessment of the results. The workflow was validated using a spiked dilution series and naturally infected samples, demonstrating that the ONT workflow achieves sensitivity and accuracy comparable to, or exceeding, that of traditional Sanger sequencing. Although PCR multiplexing was found to reduce sensitivity, limiting the immediate throughput advantages over Sanger sequencing, the proposed framework offers significant value through flexible species assignment and stringent contamination controls. Consequently, this method serves as a robust, high-resolution tool for resolving complex phytoplasma infections. [Formula: see text] Copyright © 2026 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

Abstract Geothermal systems harbor abundant novel microorganisms, representing substantial phylogenetic and functional diversity among yet‐to‐be‐cultivated bacteria with significant ecological roles. Nevertheless, research on the diversity, metabolic potential, and evolutionary history of these uncultured taxa remains limited. In this study, 19 metagenome‐assembled genomes (MAGs) were recovered from hot springs in Tengchong, Yunnan Province. Phylogenetic analyses indicate that these MAGs form sister lineages to an existing clade, leading to their proposed classification within three novel phyla: Yinglongibacterota , Xuanmingibacterota , and Gonggongibacterota , named after figures from the traditional Chinese legend, Classics of Mountains and Seas. Sequence analysis of both hot spring‐derived and published MAGs reveals that all three phyla exhibit similar facultative anaerobic lifestyles. Multiple substrates, including cellulose, glucan, pectin, and xylan, can be degraded by these phyla, especially Gonggongibacterota , suggesting their critical role in converting complex plant matter into more readily degradable substrates and contributing to sugar fermentation. Furthermore, the ability of these phyla to reduce selenite facilitates environmental detoxification. Notably, gene loss events have resulted in widespread auxotrophy for heme and biotin in these lineages, which may indicate microbial cross‐feeding in the community as an adaptation strategy. These pioneering results provide a comprehensive overview of the taxonomic and metabolic characteristics of the three novel phyla, offering insights into their ecological significance and guiding future cultivation efforts.