Publications

4866

Early and accurate detection of ‘Candidatus Phytoplasma pruni’ is essential for the control of X-disease in Prunus species, but this is made difficult by pathogen biology and the ability to consistently recover nucleic acids, both of which increase the likelihood of false negatives. Here we examined the latter, using high- and low-titer infected sweet cherry (P. avium) samples to compare three tissue disruption methods, four nucleic acid extraction chemistries, and four PCR chemistries. Results showed that method selection had minimal impact on high-titer samples but significantly affected low-titer detection. Bead-beating disruption consistently outperformed rotary and manual grinding, yielding lower Cq values and more reliable amplification. Among extraction methods, CTAB-based protocols provided the highest nucleic acid recovery and detection sensitivity, particularly when paired with bead beating. Column- and magnetic bead–based kits performed less consistently, especially with low-titer samples, where false negatives were frequent. PCR chemistry also influenced results, although RT-qPCR did not significantly improve sensitivity over qPCR. Importantly, commonly used host internal controls failed to reliably indicate pathogen detection success, as high-abundance targets masked poor extraction efficiency. Cumulatively these data suggest the need for optimizing diagnostic protocols to ensure capture of low-concentration pathogens to avoid false negatives.

Abstract Bacterial symbionts were shown to have diverse but significant impacts on the fitness of their host in insects, but little is known on the symbionts of spiders and their interactions with their hosts. Here, we assembled and investigated the circular 575 kb genome of Candidatus Argiopiplasma dusa, a novel bacterial symbiont of the wasp spider Argiope bruennichi. Phylogenomic analysis placed this species within the phylum Mycoplasmatota, in a poorly characterized clade that may represent a new order-level lineage or affiliate with the Mycoplasmatales order. With 559 predicted genes, the genome is relatively small compared to other Mycoplasmatota genomes (on average 969 genes) and has a low GC content of ∼24%. While the genome encodes genes for proteins involved in glycolysis and fermentative acetate production, it revealed minimal biosynthetic capabilities with pathways for nucleotide, amino acid and vitamin biosynthesis being absent in Ca. Argiopiplasma dusa. This suggests a metabolic dependence and an endosymbiotic lifestyle within the spider host. The symbiont was detected in A. bruennichi populations across the distribution range of the spider but appears to be absent in certain populations.

Abstract Synthetic endosymbiosis, the deliberate introduction of bacteria into eukaryotic host cells, provides a tractable model for how endosymbiotic relationships become established, and a route to cells with new metabolic capabilities that never evolved naturally in that lineage. Current methods for introducing bacteria into mammalian cells rely on microinjection, engineered invasion, listeriolysin-mediated phagosomal escape, or naturally permissive hosts. These methods are invasive, low-throughput, or costly, and most require the prospective endosymbiont to be genetically tractable, which excludes endosymbiotic bacteria which are of great interest as potential organelle precursors. Here we describe a protocol based on polyethylene glycol (PEG), long used to fuse mammalian cells, that delivers Escherichia coli into HeLa cells without genetic modification of either partner or the use of specialised equipment. We optimised the governing parameters in two stages: first, bacterial density, gentamicin concentration, and PEG molecular weight and concentration were optimised; then, medium composition and the timing of fusion and recovery. Across four parameters screened, 10% PEG-3350 applied for 4 min to HeLa cells 12 h after seeding, followed by 4 h in gentamicin-containing medium, gave the most consistent delivery. Under these conditions, confocal microscopy and z-stack reconstruction detected mCherry-tagged E. coli inside HeLa cells. Applying the same protocol to Candidatus Kinetoplastibacterium crithidii, a naturally occurring β-proteobacterial endosymbiont of the trypanosomatid Angomonas deanei , we observed the endosymbiont inside the HeLa cells. This protocol offers an accessible entry point for constructing artificial endosymbioses.

Marine bryozoans harbor diverse microbial communities, but not much is known about specificity of bacterial symbionts towards different hosts. We identified potential symbionts and described overall microbial communities of three marine bryozoans, Aquiloniella scabra, Rhamphostomella bilaminata (Gymnolaemata: Cheilostomatida), and Patinella verrucaria (Stenolaemata: Cyclostomatida), revealing that each species maintains a distinct microbial assemblage that differs both from the surrounding environmental microbiome and from other hosts. Previously, the bryostatin-producing gammaproteobacterial genus “ Candidatus Endobugula” was known from only two bryozoan species in the family Bugulidae. Here, we report its first detection in two more cheilostome bryozoans, A. scabra and R. bilaminata , expanding its known host range to two more families.

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 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.

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.

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.