Publications

4995

Rapid and accurate diagnosis of ‘ Candidatus Phytoplasma’ is critical for disease management but remains challenging due to the pathogen's unculturable nature, low titers, uneven distribution, and genetic complexity. Although current official guidelines provide a highly adaptable taxonomic foundation for species assignment, translating these broad recommendations into standardized routine diagnostics remains difficult. This study implemented the established species assignment guidelines by developing a structured, proof-of-concept diagnostic framework utilizing a multilocus sequence analysis workflow coupled with Oxford Nanopore Technologies (ONT) sequencing. The developed workflow targets four barcodes—16S, secA , rplV , and tufB —and replaces fixed demarcation criteria with a best-match principle for non-16S barcodes. Furthermore, the study offers guidance for barcode selection and challenges the use of 16S rRNA sequencing as a sole screening assay. Moreover, a read-based quantification system incorporating control thresholds was implemented to provide an objective and reliable assessment of the results. The workflow was validated using a spiked dilution series and naturally infected samples, demonstrating that the ONT workflow achieves sensitivity and accuracy comparable to, or exceeding, that of traditional Sanger sequencing. Although PCR multiplexing was found to reduce sensitivity, limiting the immediate throughput advantages over Sanger sequencing, the proposed framework offers significant value through flexible species assignment and stringent contamination controls. Consequently, this method serves as a robust, high-resolution tool for resolving complex phytoplasma infections. Copyright © 2026 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

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 .

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.