Publications

4874

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.

A novel phytoplasma species, ‘ Candidatus Phytoplasma zeae’, is proposed based on ecological distinctiveness, vector specificity, whole-genome comparisons and community consensus. This phytoplasma is associated with maize bushy stunt (MBS) disease in corn ( Zea mays ) and is transmitted exclusively by Dalbulus maidis and Dalbulus elimatus , two leafhopper species endemic to the Americas that has been reported in Brazil, Colombia, Mexico, Peru and several U.S. states. Here, we sequenced and assembled the genome of MBS phytoplasma (MBSP) strains from Brazil and the USA to describe and propose this new species. Although the 16S rRNA gene sequence of the proposed reference strain, MBSP-BR RS , shares >99% identity with that of ‘ Candidatus Phytoplasma asteris’, key nucleotide polymorphisms distinguish ‘ Ca . P. zeae’ from other 16SrI-related phytoplasma species. Genome-wide average nucleotide identity between ‘ Ca . P. zeae’ and all available ‘ Ca . P. asteris’-related strains genomes was between 92.6 and 98.4%. Comparative genomic analyses revealed unique gene clusters in ‘ Ca . P. zeae’ associated with amino acid transport, defence mechanisms and protein turnover, which may contribute to its specialization in corn. The ecological profile of ‘ Ca . P. zeae’, including its narrow host and vector range and restricted geographic distribution, supports its recognition as a novel species under Rule c of the IRPCM guidelines. The designation ‘ Ca . P. zeae’ is, therefore, proposed by members of the research community who have studied this pathogen for over a decade, with the MBSP-Brazil RS strain serving as the reference.

ABSTRACT Plant microbiomes are a rich source of secondary metabolites, often with pathogen- or disease-suppressive traits. Huanglongbing (HLB) is a serious disease of citrus for which there is currently no cure. Here, we tested the hypothesis that bacteria native to the citrus microbiome may possess inhibitory activity against the associated causal agent of HLB, Candidatus Liberibacter asiaticus ( C Las). We coupled a robotic microfluidic isolation platform with a high-throughput antimicrobial screening assay using Liberibacter crescens , a culturable surrogate for the unculturable C Las, to identify isolates with anti- Liberibacter activity. We identified 35 bacterial isolates with activity using this pipeline. One strongly inhibitory citrus root isolate, Pseudomonas chlororaphis (SRAM11), was selected for bioassay-guided fractionation. We determined a significant driver of in vitro Liberibacter inhibition was attributed to the production of phenazine-1-carboxamide (PCN) by P. chlororaphis . We also demonstrated significant anti- C Las activity of PCN, comparable to that of oxytetracycline, using an ex vivo citrus hairy root assay. P. chlororaphis and phenazines have been implicated in successful biocontrol of other diseases and represent a new tool with possible application to HLB mitigation. IMPORTANCE Huanglongbing (HLB) is a devastating citrus disease that causes millions of dollars in crop loss each year in the United States alone. In this study, we mined the citrus microbiome for bacteria that produce antimicrobials with suppressive activity against Candidatus Liberibacter asiaticus, the causal agent of HLB. We demonstrated that the citrus microbiome is a rich source of microorganisms that inhibit Liberibacter spp. We selected a Pseudomonas chlororaphis strain for further characterization and assigned anti- Liberibacter activity to the production of phenazine-1-carboxamide. Phenazine-producing P. chlororaphis strains are known biocontrols in other pathosystems, and this study highlights their potential for mitigation of HLB.

Enhanced biological phosphorus removal (EBPR) relies on polyphosphate-accumulating organisms that experience dynamic fluctuations in redox conditions and substrate availability. How these organisms regulate metabolism across molecular layers under cyclic operation remains incompletely understood. Here, we investigated molecular regulation in a long-term, stable aerobic granular sludge system dominated by Candidatus Accumulibacter. We combined metagenomics, time-resolved metatranscriptomics, and matched metaproteomics across three consecutive operational cycles. Genome-resolved metagenomics revealed an Accumulibacter-dominated community encoding canonical EBPR functions. Time-resolved metatranscriptomics revealed extensive and reproducible transcriptional dynamics affecting hundreds of genes. These dynamics did not align with discrete EBPR phases or instantaneous environmental transitions, but instead unfolded gradually over the cycle. Despite pronounced transcript-level heterogeneity, aggregation at the functional level revealed strong consolidation, with only a limited number of KEGG orthologs exhibiting coherent temporal regulation. Quantitative metaproteomics revealed a stable proteome across EBPR cycles, with no individual proteins or functional categories showing significant differential abundance despite direct transcript-protein mapping and time-matched sampling. This indicated that transcriptional dynamics did not translate into measurable changes in protein pool size on the time scale of individual cycles, consistent with slow, growth-coupled proteome turnover relative to cycle duration. Together, these results support a hierarchical model of regulation in Candidatus Accumulibacter, in which dynamic transcriptional responses are buffered at both the functional and protein levels. This organization provides a plausible basis for stable EBPR performance under predictable cyclic operation and demonstrates how time-resolved multi-omics can be used to resolve regulatory dynamics across molecular layers in engineered microbial ecosystems.

Abstract In northeastern Argentina, wild canids such as the pampas fox ( Lycalopex gymnocercus ) and the crab‐eating fox ( Cerdocyon thous ) coexist with domestic dogs ( Canis lupus familiaris ) across rural and peri‐urban landscapes, creating opportunities for tick and pathogen exchange. Characterizing tick assemblages and associated pathogens in these hosts is essential to assess potential public health risks at the wildlife–domestic interface. Ticks were collected opportunistically from domestic dogs and road‐killed foxes near two protected areas in Corrientes Province. Specimens were identified using morphological and molecular approaches and screened for Rickettsia spp. by PCR. A total of 30 adult ticks were obtained from 12 dogs and 8 foxes, representing four species: Amblyomma tigrinum , A. ovale , A. dubitatum and Rhipicephalus sanguineus . Amblyomma tigrinum was the most frequent species (50%). Seven individual ticks were confirmed as Candidatus Rickettsia andeanae in A. tigrinum , while one pooled sample was positive by screening only and was not confirmed. These findings expand the known geographic distribution of Ca. R. andeanae and provide molecular evidence of its circulation in ticks collected from both wild and domestic canids in northeastern Argentina. Our results highlight the importance of these hosts as sentinels for tick‐borne pathogens and underscore the need for integrated One Health surveillance to better anticipate emerging potential risks in interface areas.

Huanglongbing (HLB) is one of the most devastating diseases of citrus nowadays. The etiological agent is a fastidious and non-culturable bacterium known as Candidatus Liberibacter asiaticus ( C Las). C Las is a phloem-restricted pathogen, which makes the efficient delivery of antimicrobial compounds to control it a major challenge for citriculture. Therefore, the development of formulations capable of penetrating plant barriers and reducing the bacterial population within the phloem is critical for the control of HLB. Here we report on the development of a novel formulation loaded with oregano essential oil, hexyl gallate and eugenol, described in the literature as a transdermal permeation enhancer. The most effective formula was selected in vitro against bacterial surrogates ( Rhizobium tropici and Rhizobium phaseoli ), displaying bactericidal concentrations of 0.25 and 0.5% (v/v), respectively, of bactericidal concentration. The selected formulation was characterized as a near-Newtonian fluid, with 98.5% of particles measuring around 50 nm, a Polydispersity Index of 0.31, and zeta potential of -17.5 mV. Furthermore, Scanning electron microscopy revealed the presence of nanoparticles, alongside microscopic aggregates. The formulation maintained its bactericidal activity over 120-day storage period. During the root-drenching assays, the formulation significantly reduced overall C Las titres by 1.29 and 2.46 log 10 over a period of 300 days, in two independent experiments. Furthermore, ecotoxicological assessments revealed no acute toxicity in Tenebrio molitor or significant effects in the survival of Melipona scutellaris under the tested conditions. Our findings demonstrate the potential of this non-synthetic formulation as an effective and safe alternative for HLB management, without disrupting non-target organisms.