Publications

4446

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 ‘Candidatus Mycoplasma haemolamae’ (CMhl) is a hemotropic bacterium infecting South American Camelids (SAC), whose epidemiology and clinical significance are still not fully elucidated. This study investigated CMhl by qPCR in blood samples of alpacas ( Vicugna pacos ) and llamas ( Lama glama ) in Italy in 2021–2024, characterizing CMhl haplotypes based on partial 16 S rRNA sequences and defining haematological findings in a subset of CMhl-infected animals. Statistical analysis was performed to detect potential risk factors associated with CMhl positivity. Out of 206 animals (200 alpacas and 6 llamas), CMhl was detected by qPCR in 88 (42.7%) animals. CMhl haplotype #1 and #2 were detected in 42.2% and 0.5% of the animals, respectively. Binary logistic regression analysis showed that higher CMhl positivity was significantly associated with larger herd size ( P  = 0.011), Northern Italy area of origin ( P  = 0.031) and warmer seasons ( P  = 0.003). The detection of two CMhl haplotypes confirms the high diffusion of CMhl in SAC farmed in Italy. Results confirm that CMhl usually causes subclinical infections, suggesting that antimicrobial treatment is likely not necessary based on qPCR positivity alone. Results focusing on the association of CMhl positivity with warm temperatures and large herd size suggest the need of further studies on the genetic features, epidemiology and immune response in CMhl-positive SAC from Italy, with emphasis on understanding potential vector transmission.

The Clostridium estertheticum complex (CEC) consists of closely related bacterial species that are mostly isolated from meat processing environment. Genome mining studies have recently established CEC as a source of class I bacteriocins. However, up until now, class II bacteriocins have not been reported from CEC. In the present study, we determined the presence of class II bacteriocin biosynthetic clusters in 33 CEC genomes through genome mining followed by bacteriocin production through heterologous expression in Escherichia coli . Six biosynthetic gene clusters belonging to class IIa ( n  = 1), IIb ( n  = 2), IId ( n  = 2) and an undefined class containing three precursor peptides were identified in six different CEC strains. Using molecular biology, we developed dedicated expression vectors for the class IIa bacteriocin, clesteriocin A. Its precursor peptide, CleA, and protease, CleB150, were initially expressed in insoluble form in E. coli . Through a systematic analysis of suitable solubility enhancing protein tags, both CleA and CleB150 were expressed in significant amounts of soluble fractions using tandem tags derived from Small Ubiquitin-like Modifier, superfolder Green fluorescent protein, Maltose binding protein and N-utilization substance. Mature clesteriocin A was obtained following in vitro maturation reactions between the tagged CleA and CleB150. The novel bacteriocin displayed antimicrobial activity against Listeria monocytogenes , which was mediated by the mptC gene. By combining genome mining and molecular biology, we have shown CEC is a source of novel class II bacteriocins that have potential for application as food biopreservatives.

Abstract Multicellular magnetotactic prokaryotes represent a unique group of obligately marine multicellular bacteria known for their ability to navigate along magnetic field lines thanks to ferrimagnetic nanocrystals. To date, two distinct spherical and ellipsoidal morphotypes have been described, typically ranging from 3 to 6 μm in diameter and comprising approximately 50 cells of the same species. Although widespread in highly reduced marine sediments, they are represented by solely three genera clustering into a monophyletic group within the Desulfobacterota. In this study, we report a third morphotype in reduced sediments of the Mediterranean Sea in Carry-le-Rouet, France, that is approximately 30 times more voluminous than any previously described form. Because their large size, we designated these multicellular bacteria as “giant” and explored their cell ultrastructure, ecological niche and physiology using magnetic enrichment and a combination of microscopy techniques and single-consortium genomics. Transmission electron microscopy and confocal microscopy images of several individual consortia revealed that they contain an average of 130 cells, each producing over 100 greigite magnetosomes arranged to optimize the overall magnetic moment. Phylogenomic analyses positioned giant multicellular magnetotactic prokaryotes, together with other morphotypes, in a previously undescribed genus and species within the Candidatus Magnetomoraceae family, named Magnetogigantoglobus mediterraneus. Although genetically divergent with a different ultrastructure, all multicellular magnetotactic prokaryotes seem to rely on sulfate reduction coupled to heterotrophy or autotrophy. We further discuss the significance of these findings in the context of the evolutionary history of multicellularity and magnetotaxis in prokaryotes.

Abstract BACKGROUND The Asian citrus psyllid, Diaphorina citri , vectors Candidatus Liberibacter asiaticus ( C Las) that causes citrus greening disease, which has devastated global citrus production. Current management primarily relies on the application of synthetic chemical insecticides, resulting in the evolution of resistance among D . citri populations. C Las is ingested by D . citri while feeding on infected plants and subsequently interacts with the gut epithelial surface to establish infection. To determine the functional significance of abundant gut surface proteins in D. citri during C Las uptake, we used RNA interference (RNAi) to silence nine key gut surface protein genes. RESULTS Following membrane feeding on double‐stranded RNAs (dsRNAs), transcript levels were reduced by 20–51% in adults and 28–50% in nymphs at 72 h, with higher knockdown efficiencies of 41–63% in adults and 30–81% in nymphs by 120 h. Some dsRNA treatments increased D. citri mortality by approximately 20% over the control treatment, and two treatments reduced honeydew production indicative of reduced feeding consistent with disrupted gut function. Silencing of several individual proteins, including alkaline phosphatase, cadherin, cluster of calcium‐transporting ATPase, croquemort and monocarboxylate transporter, reduced C Las uptake in nymphs and/or adults. CONCLUSION Silencing of abundant gut surface proteins in D. citri limited C Las uptake, establishing a functional basis for RNAi‐mediated suppression of pathogen uptake. While additional studies are needed to determine specific molecular interactions, these results identify gut surface proteins as promising targets for RNAi‐based interventions to reduce C Las uptake by psyllids. © 2026 Society of Chemical Industry.

Pear decline, caused by 'Candidatus Phytoplasma pyri', has emerged in Chilean pear orchards in recent years. While several Cacopsylla species are potential vectors of 'Ca. P. pyri', the disease's full epidemiological cycle remains uncertain. Cacopsylla bidens, present in Chilean orchards, has recently been reported as a vector. This study conducted year-long surveys in two commercial pear orchards across different Chilean regions, capturing C. bidens in zones with 'Ca. P. pyri'-infected pear trees. All developmental stages were collected, with peak abundances occurring in March and April. Outside the study zones, C. bidens were found in pine trees but not in adjacent cultivated areas. Two seasonal morphotypes, summer and winter forms, were identified. Molecular analysis detected 'Ca. P. pyri' in a high proportion of insects, with maximum infection rates in March and April. These findings advance our understanding of 'Ca. P. pyri' spatial and temporal dynamics and its potential role in 'Ca. P. pyri' spreading under Chilean field conditions.

In 2022 and 2024, field surveys were conducted in the Columbia Basin of Washington State, USA, for pathogens transmitted by the beet leafhopper, Circulifer tenellus, including beet curly top virus (BCTV), ‘Candidatus Phytoplasma trifolii’ (CPt), and Spiroplasma citri (S. citri). For these pathogens, an association of symptoms with a causal agent is often complicated by co-infections and the inability to culture CPt (Hu 2021; Rivedal et al. 2022, 2024; Schoener & Wang 2023). In 2022, 35 hemp (Cannabis sativa) plants were observed at the Washington State University (WSU) Research Station in Othello, WA, with typical symptoms of BCTV (a finding published by Jarugula et al. 2023) and we further tested these samples for C. tenellus-associated bacteria. In 2024, 23 and 15 hemp plants observed in Prosser, WA, from the WSU Irrigated Agriculture Research Center and WSU Roza Farm, respectively, with symptoms of CPt infection, including stem fasciation and purpling. Leaf tissue from each plant was ground in liquid nitrogen, DNA extracted using the DNeasy Plant Mini Kit (Qiagen, Inc.), and a multiplex real-time PCR protocol used to detect the C. tenellus-associated pathogens (Swisher Grimm et al. 2023). CPt was identified in 8.6% and 65.8% of plants in 2022 and 2024, respectively, while S. citri was not detected in 2022 but was found in 15.8% of plants in 2024. Co-infections were common. Nested PCR (primers P1/P7 and R16F2n/R16R2, Crosslin et al. 2006) and conventional PCR (primers Trif-SecY-163F, 5ʹ-AGCAGCTAAAAAAGTTAGAAAAAACCTC-3ʹ/Trif-SecY-1040R, 5ʹ-AAATCTAGCGAAAATGATTTTTTGTTTTCA-3ʹ) targeted the CPt 16S rRNA and secY genes, respectively. S. citri infection was confirmed by PCR targeting the spiralin gene (primers SpiralinF/R, Yokomi et al. 2008). All targets were amplified using PrimeSTAR HS DNA polymerase (Takara Bio Inc.). Thermal cycle conditions for CPt 16S rRNA and S. citri spiralin genes consisted of 30 or 40 cycles, respectively, at 98°C for 10 s, 55°C for 15 s, and 72°C for 10 s, followed by a final elongation at 72°C for 5 min. Conditions for CPt secY consisted of 35 cycles at 98°C for 15 s, 55°C for 30 s, and 72°C for 10 s, followed by a final elongation at 72°C for 5 min. Amplicons of size 1,250 bp, 877 bp, and 675 bp for the 16S rRNA, secY, and spiralin genes, respectively, were visualized on an agarose gel stained with GelRed (Sigma-Aldrich) or Ethidium Bromide. For CPt, sequencing of 16S rRNA from seven infected samples (2 from 2022, 5 from 2024) revealed identical sequences (GenBank Accession PV983691) with 100% identity to CPt in U.S. hemp (OQ597521), and sequencing of secY in one sample from 2022 and one from 2024 revealed identical sequences (PX725980), with 100% identity to CPt in U.S. periwinkle (GU004317). For S. citri, two sequenced samples had identical spiralin sequences (PV955037), matching 100% and 99.85% with S. citri found in Oregon cabbage (PV099668) and hemp (OQ969984), respectively. This is the first confirmed report of CPt and S. citri in C. sativa in Washington state. These findings highlight the need to evaluate effects of mollicutes on hemp and underscore the need to develop integrated pest management strategies to reduce vector transmission. In addition, these findings suggest that hemp could serve as a reservoir of the C. tenellus-transmitted pathogens, leading to higher pathogen prevalence across the region, and negatively impacting economically important vegetable and seed crops grown in Washington state that are susceptible to these pathogens.