Publications

4861

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.

The family Gallionellaceae includes neutrophilic, aerobic, iron-oxidizing bacteria, many of which remain uncultured and known only from metagenomic data. In this study, two high-quality Gallionellaceae genomes were assembled from a ferruginous spring in the Voronezh region, Russia. Phylogenetic analysis based on 120 concatenated proteins, ANI (average nucleotide identity), and AAI (average amino acid identity) indicates that BG-057 belongs to the genus Gallionella and BG-364 to the genus 39-52-133. We propose the names “Candidatus Gallionella aquifuscii” sp. nov. for BG-057 and “Candidatus Dubininella ochracea” gen. nov., sp. nov. for BG-364. Both genomes encode genes for the Calvin-Benson-Bassham (CBB) cycle. Both are potentially capable of aerobic respiration due to the presence of electron transfer chain components. Both genomes encode the iron oxidase Cyc2, and BG-057 also encodes MtoA. BG-057 is potentially capable of lithotrophic growth in the presence of H2 and H2S. BG-364 is potentially capable of growth via Mn (II) oxidation; arsenite oxidation; the direct oxidation of thiosulfate; H2S, elemental sulfur, and sulfite oxidation. BG-364 encodes denitrification genes (narGHI, nirS, norBC). BG-057 possesses systems for the biosynthesis and export of siderophores, while BG-364 lacks these systems but retains Ton-dependent receptors, suggesting the use of exogenous siderophores.

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.

Abstract Purpose The objective of this study was to characterize Bradyrhizobium strains currently recommended as inoculants for Arachis pintoi in Brazil using comparative genomic and symbiotic analyses. In addition, this study aimed to expand current knowledge on the diversity, evolution, and bacterial symbiotic traits of Arachis -associated Bradyrhizobium while providing robust taxonomic evidence to support the description of a novel Bradyrhizobium species isolated from root nodules of Arachis . Methods Phylogenetic reconstruction was performed based on the 16S rRNA gene and by phylogenomics using 116 single- copy core gene sequences. Average Nucleotide Identity (ANI) analyses were conducted to evaluate the genomic relatedness of strains BR 1432ᵀ, BR 1433 and BR 14999 to 1,301 reference Bradyrhizobium genomes, including all available type strain strains. The results were validated using digital DNA–DNA hybridization (dDDH). Additional analyses included phylogenetic reconstruction of symbiotic genes ( nodC and nifH ) and, assessment of the denitrification pathway, and phenotypic characterization. Results Phylogenetic analyses based on 16S rRNA gene sequences and whole-genome data indicated that strains BR 1432ᵀ and BR 14999 form a distinct lineage within the genus Bradyrhizobium , clearly separated from BR 1433, which clustered with Bradyrhizobium brasilense . ANI and dDDH values between BR 1432ᵀ and BR 14999 and their closest related type strains, including B. zhanjiangense LMG 29279ᵀ, were below the species delineation thresholds of 96% and 70%, respectively, supporting the proposal of a novel species. In addition, the publicly available RefSeq genomes Bradyrhizobium sp. DN5 and Rc2d were found to be closely related to BR 1432ᵀ. Phylogenetic analyses of the nodC and nifH genes revealed distinct evolutionary patterns, while genomic analyses showed that the proposed novel species harbors a complete denitrification pathway, unlike strain BR 1433. Furthermore, phenotypic characterization revealed differences in carbon source utilization and enzymatic activities. Conclusion Based on genomic, phylogenetic, and phenotypic evidence, strain BR 1433 was identified as a member of B. brasilense , whereas strains BR 1432ᵀ (= UCCC 277ᵀ = LMG 34120ᵀ = CCP577ᵀ), BR 14999, DN5 and Rc2d represent a novel species within the genus Bradyrhizobium , for which the name Bradyrhizobium boddeyi sp. nov. is proposed.

Huanglongbing (aka: citrus greening) associated with ‘Candidatus Liberibacter asiaticus’ (CLas) is one of the most impactful citrus diseases throughout the world. The aim of this project was to support the sensitive and rapid detection of CLas to prevent the continued spread and destruction of US citrus crops by this psyllid vector-borne disease. Two multicopy targets specific to CLas were selected for assay development: the five-copy nrdB (ribonucleotide reductase-RNR) gene and a four-copy locus 4cp (4CP). Published RNR RPA primers were used to develop a one-pot RPA-CRISPR assay, and RPA primers for 4CP were designed. crRNAs for both RNR- and 4CP-RPA-CRISPR/Cas assays were designed, evaluated through preliminary testing, and the best candidate primers and crRNA combinations were selected. The 4CP assay was selected and subjected to a battery of validation tests and determined fit-for-purpose. A field extraction protocol was also developed using commercial reagents, syringes and filters to bring kit sensitivity to the field. When combined with the assay, the complete workflow was able to detect CLas in infected citrus, down to a single infected leaf per subsample; the field workflow showed similar sensitivity to the laboratory-based workflow. While the extraction and assay proved useful, particularly for symptomatic leaves, its performance with asymptomatic leaves for early detection was not evaluated. The complexity of the assay in its current form hinders widespread practical applicability. In the future, revalidation of lyophilized reagents in a convenient kit can be performed for practical deployment; preliminary testing has shown success.