Publications

4851

Abstract Background ‘ Candidatus Liberibacter solanacearum’ (Lso) is a phloem-limited bacterial pathogen causing significant diseases in solanaceous crops. In the United States, haplotypes A and B are transmitted by the potato psyllid Bactericera cockerelli . We previously identified differences in their acquisition and transmission between adults and nymphs. The present study characterized the dynamics of LsoA and LsoB acquisition and transmission by nymphs and examined the transcriptional responses of the nymphal gut upon their acquisition. Methods and results Nymphs were exposed to LsoA- or LsoB-infected plants for 1, 3, 5, or 7 days to measure the bacterial accumulation and for 8 days to assess the transmission efficiency following sequential inoculation of tomato plants. Quantitative PCR showed that LsoB accumulated to higher levels than LsoA after 3 days of acquisition. Following the sequential inoculation, LsoB was transmitted earlier than LsoA indicating a shorter latency period. RNA-seq analysis of the guts following a 1- and 5-day acquisition access periods revealed a greater transcriptional regulation at 5 days than at 1 day. Furthermore, the responses were haplotype-specific: LsoA primarily affected genes involved in protein translation, ER stress, and cell cycle regulation, whereas LsoB regulated genes involved in autophagy, apoptosis, and immune pathways. Conclusions This study revealed haplotype-specific gene regulation potentially leading to LsoB being transmitted more efficiently by psyllid nymphs.

Abstract Background The Toll signaling pathway is a key component of insect innate immunity, yet its role in hemipteran vectors and their interactions with plant pathogens remains poorly understood. The potato psyllid ( Bactericera cockerelli ), an important agricultural pest in the USA, transmits two haplotypes of ‘Candidatus Liberibacter solanacearum ’ (LsoA and LsoB) that cause severe diseases in solanaceous crops. Methods and Results We characterized Toll pathway components in the B. cockerelli genome and examined their transcriptional responses during pathogen acquisition. Six Toll receptors and ten pathway-associated genes were identified in the psyllid genome. Gene expression was evaluated using RT-qPCR following exposure to the entomopathogenic fungus Beauveria bassiana (Bb-490) and infection with Lso haplotypes. The transcription of five Toll receptors was significantly induced in response to B. bassiana , indicating their potential role in antifungal defense. In contrast, Lso infection elicited haplotype-specific transcriptional responses: two Toll receptors (Toll like-1 and Toll-7) were up-regulated during LsoB acquisition, whereas only Toll-7 responded to LsoA. In addition, Spätzle-3 was induced by LsoB, while Spätzle was induced by LsoA. Persistent Lso infection did not significantly alter the transcriptional expression of Toll receptors. Conclusions These findings provide new insights into immune regulation in B. cockerelli and highlight the potential of targeting Toll-mediated responses and fungal biocontrol strategies for sustainable 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 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 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.