Publications

4943

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 ‘ Candidatus Phytoplasma solani’ is a widespread and epidemiologically important phytoplasma in Europe with a broad host range, including sugar beet, potato and grapevine. In sugar beet, the recently emerging 16SrXII‐P subgroup of ‘ Ca . P. solani' frequently co‐occurs with the phloem‐associated bacterium ‘ Candidatus Arsenophonus phytopathogenicus’, which is considered the primary causal agent of the ‘syndrome des basses richesses’ (SBR), a disease that significantly affects yield and sugar content in central Europe. Currently, diagnosis and differentiation of ‘ Ca . P. solani' rely on real‐time quantitative PCR (qPCR) and nested PCR, which are labour‐intensive, costly and require complex instrumentation. Therefore, we developed a loop‐mediated isothermal amplification (LAMP) assay for specific detection of 16SrXII‐P phytoplasma that is suitable for rapid laboratory use and potential on‐site testing using crude plant and insect extracts. The new LAMP assay targets the stamp gene of 16SrXII‐P and was evaluated together with a published LAMP assay targeting the secA gene of ‘ Ca . P. solani' 16SrXII‐A. This combination enabled reliable detection and discrimination of 16SrXII‐P and 16SrXII‐A phytoplasma in sugar beet tissues and in the vector Pentastiridius leporinus . The 16SrXII‐P‐specific LAMP assay showed high analytical specificity and showed comparable performance to a TaqMan qPCR assay in the reliable detection of positive samples. In combination with simple NaOH‐Tris and phosphate‐buffered saline (PBS)‐based crude extraction protocols, the assay provided clear positive or negative results within 30 min, making it a practical tool for surveillance of 16SrXII‐P phytoplasma and rapid differentiation between the two ‘ Ca . P. solani' subgroups infecting sugar beet.

Citrus huanglongbing (HLB) is recognized as the most destructive bacterial disease affecting citrus. Among the causal agents, ‘ Candidatus Liberibacter asiaticus’ (Las) is the predominant species causing HLB epidemics worldwide. Detecting this phloem-limited bacterium at early or low-titer stages and assessing its cellular activity remain major challenges for HLB research and management. To address these limitations, we developed a one-step reverse transcription quantitative PCR (RT-qPCR) assay for highly sensitive Las detection using total nucleic acids and the established Li 16S rRNA/rDNA primer–probe set. This method simultaneously amplifies both Las 16S rRNA and rDNA in a single reaction, increasing sensitivity by up to 500-fold compared with qPCR, which targets only 16S rDNA. The improved sensitivity reduced false negatives, expanded sampling capacity, and enabled detection of low-titer infections previously undetectable. In addition, the higher abundance of 16S rRNA generated a consistent Ct gap between RT-qPCR and qPCR, which we used to estimate relative Las cell activity in vivo in host insects and plants and to identify the most effective inoculum sources. This comparative detection strategy was further applied to evaluate inoculum potential, to monitor changes in relative cell activity during in vitro cultures, and to assess antimicrobial treatments targeting Las. The persistent detection of long-lasting, low-titer Las infections underscores the complex biology of the HLB causal agents and highlights ongoing challenges for effective disease management.

The phloem-colonizing bacterial pathogen ‘ Candidatus Liberibacter asiaticus’ (CLas) causes the devastating citrus huanglongbing (HLB). Despite the important roles of roots, how CLas affects roots remains largely unknown. Here, we dissected host responses in leaf and root at different time points after CLas inoculation. Our data showed that CLas triggers less reactive oxygen species (ROS) accumulation and phloem callose deposition, which are known to contribute to HLB symptom development in leaves, in roots than in leaves. Because photosynthetically active chloroplasts are abundant in leaf tissues, but absent in roots, and serve as major hubs of pathogen-induced ROS production, elevated chloroplast-associated ROS likely contributes to the greater ROS accumulation observed in leaves during CLas infection. qRT-PCR analyses in roots and leaves, together with root RNA-seq analysis and comparison with previously reported leaf transcriptomic datasets, showed global induction of immune-related pathways in both roots and leaves, whereas photosynthesis-related pathways were only downregulated in leaves, but not in roots, by CLas infection. The differential expression pattern related to immunity and photosynthesis in the leaf and root tissues suggests that chloroplasts might be involved in the differences in ROS accumulation and callose deposition. Root cell death was observed in HLB-symptomatic trees regardless of the CLas infection status of roots. Significantly more starch accumulation was first observed in CLas-positive leaves than in healthy controls, followed by starch depletion in CLas-positive roots later, suggesting a causative relationship between them. Taken together, root cell death caused by CLas infection may be caused by carbon starvation rather than via a similar mechanism as in leaves. Our data demonstrate that chloroplast-associated ROS accumulation plays a critical role in the tissue-specific host responses to CLas infection and provide a framework for future investigations into the mechanisms underlying HLB pathogenesis. Copyright © 2026 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

ABSTRACT Huanglongbing (HLB) is a devastating and incurable citrus disease associated with the gram-negative, phloem-limited, and unculturable bacterium “ Candidatus Liberibacter asiaticus” ( C Las), transmitted by the psyllid Diaphorina citri . Our goal was to identify high-performing microbial candidates from a previously established citrus-associated microbial collection for the management of HLB and associated root pathogens. Candidate selection was based on bioactivity, genomic, and metabolomic profiles. Eighteen Bacillus isolates were screened for antagonistic activity against Liberibacter crescens , C Las, and citrus-associated Phytophthora and Fusarium spp. Phylogenetic analysis grouped the isolates into four species groups: Bacillus velezensis , B. subtilis , B. pumilus , and B. cereus . Among these, B. velezensis exhibited the strongest and broadest inhibitory activity, followed by B. subtilis . This enhanced antagonistic potential is likely associated with the greater number and diversity of biosynthetic gene clusters (BGCs) identified. Notably, C Las-inhibitory isolates included B. velezensis (CB0021 and CB0909) and B. altitudinis (CB0086). Bacilysin and surfactin BGCs were conserved across all C Las-inhibitory isolates, suggesting a potential role in C Las suppression. Accordingly, surfactins were detected in bioactive fractions of B. velezensis (CB0027 and CB0909) and B. subtilis (CB0893), supporting their likely contribution to bioactivity and inhibiting L. crescens growth in vitro , highlighting their potential as candidate compounds for C Las management. Overall, this study underscores the importance of microbial discovery, screening, and characterization as an effective strategy for identifying sustainable solutions to manage HLB. IMPORTANCE Huanglongbing (HLB) is a severe disease of citrus that has altered the citrus landscape worldwide and currently has no cure. It is caused by the bacterium “ Candidatus Liberibacter asiaticus” ( C Las) and transmitted by the psyllid Diaphorina citri . Currently, the most effective control strategy for this disease relies on trunk injection of antibiotics oxytetracycline and streptomycin, and there is a need to find alternative solutions to complement or replace chemical control. Here, we aimed to select high-performing candidates from our previously established citrus-associated microbial collection to manage HLB and root-associated pathogens. We screened 18 Bacillus isolates for antagonistic activity against Liberibacter crescens , C Las, and citrus-associated Phytophthora and Fusarium spp., and we generated their genomic and metabolomic profiles. Our research centers on selecting microbial candidates for their bioactivity and antimicrobial metabolite production, aiming to develop effective plant-protective bioinoculants for mitigation of HLB.

Chokecherry ( Prunus virginiana L.) is a native shrub that is widely found across North America and is a host of ‘ Candidatus Phytoplasma pruni’ associated with X-disease of stone fruits. This pathogen is of major concern to U.S. cherry and peach growers, with the most recent and ongoing outbreak in the Pacific Northwest causing major economic harm. To investigate the role of chokecherry as a potential reservoir and source of orchard-associated strains, we surveyed wild chokecherry trees across 12 U.S. states and compared the strains present with those in commercially cultivated Prunus species. Approximately 47% of chokecherry sampled tested positive for a ‘ Ca. P. pruni’-related strain and phylogenetic analysis revealed high strain diversity, including six distinct clades and four newly identified lineages. In contrast, ‘ Ca. P. pruni’ strains isolated from cultivated cherry and peach trees exhibited lower diversity and were infected by known disease outbreak-associated strains such as Northwestern-X. These data suggest limited transmission between wild and cultivated hosts in the western United States, with patterns indicating spillover from orchards into nearby chokecherry populations rather than the reverse. However, in states east of the Rocky Mountains, chokecherry likely functions as a reservoir for orchard infections.