Publications

4912

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.

ABSTRACT Citrus greening disease, associated with the bacterium Candidatus Liberibacter asiaticus ( C Las), has severely impacted citrus yields on a global scale. C Las is vectored by the Asian citrus psyllid, Diaphorina citri Kuwayama, in a circulative and propagative manner. Extensive application of chemical insecticides for suppression of psyllid populations has resulted in widespread resistance. Despite considerable effort and expenditure, citrus greening disease continues to impair citrus production. For transmission to occur, C Las must bind and infect the psyllid vector via the gut epithelium, but little is known of the specific molecular interactions involved. By screening a phage display library, we selected ten psyllid gut binding peptides, a subset of which have sequence similarity to C Las surface proteins. Peptide binding to the surface of the psyllid gut epithelium was confirmed by identification of gut proteins bound by specific peptides. We evaluated whether these peptides could interfere with the interaction between C Las and the gut epithelium of D. citri , thereby impeding pathogen transmission. Peptide DcNy2 significantly reduced C Las acquisition and inoculation by D. citri compared to control treatments. Nymphs fed specific peptides exhibited reduced C Las acquisition and infection of both the alimentary canal and salivary glands without negatively impacting psyllid fecundity, development, or survival. These results support peptide-mediated disruption of C Las transmission by D. citri as a potential component of integrated strategies to manage citrus greening disease. IMPORTANCE Citrus greening disease, or huanglongbing, causes billions of dollars in losses and represents one of the greatest challenges facing global citrus production. Despite significant investment on multiple fronts, effective disease management strategies are lacking. This study shows for the first time that short peptides that bind to the surface of the gut of the Asian citrus psyllid vector interfere with C Las association with the gut. This critical first gut binding step is required for C Las to infect and propagate within the psyllid vector. The downstream consequence of this peptide-mediated interference is reduced acquisition and transmission of the citrus pathogen. This work provides the foundation for new transgenic or paratransgenic tools for use in the management of citrus greening disease.

Abstract The majority of marine nitrite-oxidising bacteria (NOB) belong to three uncultured genera within the Nitrospinaceae family and thus the ecophysiology of these main nitrate producing microorganisms in the ocean remains poorly resolved. Here, we report on the environmental characterization of one of these genera, which we name Candidatus Nitromaricola. A single species within this genus, Candidatus Nitromaricola pontica, comprises >95% of the nitrite-oxidising community in the permanently stratified Black Sea. This provided a unique opportunity to study this globally abundant genus in situ, across pronounced environmental gradients of oxygen and inorganic nitrogen concentrations. We show that Ca. Nitromaricola pontica is capable of oxidising nitrite at similar rates in both oxic and apparently anoxic waters, although it had a higher abundance in the more oxygenated waters of the Black Sea. The genome of Ca. Nitromaricola pontica indicates that like other NOB, it is a chemolithoautotroph, oxidising nitrite, and fixing inorganic carbon. However, Ca. Nitromaricola pontica and other members of the Ca. Nitromaricola genus lack canonical high affinity terminal oxidases found in some other NOB genera. Single-cell analyses confirmed active autotrophic growth and showed that ammonium and urea, rather than nitrite, were the main nitrogen sources for biomass production. Our findings identify Ca. Nitromaricola as a globally abundant marine NOB and highlight its cosmopolitan lifestyle.

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.

Abstract 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.