Publications

4859

Huanglongbing (HLB), caused by ‘Candidatus Liberibacter asiaticus’ (CLas), poses a major threat to citrus production worldwide and has recently emerged as a serious constraint to sweet orange cultivation in Bangladesh. Despite widespread disease occurrence, information on the genetic diversity of CLas populations in the country is lacking. This study investigated the genetic structure of CLas across major sweet orange–growing regions of Bangladesh using tandem repeat number (TRN) variation at the CLIBASIA_01645 genomic locus. Field surveys were conducted in 17 districts between 2022 and 2023, and 89 HLB-symptomatic leaf samples were collected from commercial orchards. PCR and qPCR detection confirmed CLas infection in 67 (75%) and 74 (83%) samples, respectively. Thirty-seven representative isolates were sequenced and analyzed for TRN polymorphism. Ten TRN genotypes (TRN2–TRN12, excluding TRN3) were identified, with TRN6 being the most prevalent. Bangladeshi CLas isolates were further grouped into three classes based on TRN: Class I (TRN ≤ 5), Class II (5 < TRN ≤ 10), and Class III (10 < TRN ≤ 15), of which Class II was the predominant. All Bangladeshi isolates harbored the CTTGTGT repeat motif and clustered closely with Indian isolates in haplotype network and phylogenetic analyses, while isolates carrying the AGACACA motif from other countries formed a distinct clade. These results reveal intraspecific diversity of CLas in Bangladesh and suggest regional structuring and shared ancestry with neighboring Indian populations. This study provides the first population-level genetic insight into CLas in Bangladesh and establishes a foundation for improved epidemiological understanding and disease management.

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 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.

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.

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.

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.