Publications

4794

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.

The distribution of ‘Candidatus Phytoplasma prunorum’, a phloem-limited pathogen, varies among plant tissues during the year, but knowledge about quantitative comparisons of its presence in roots and above-ground tissues across Prunus species remains limited. The variation in ‘Ca. P. prunorum’ in four Prunus species was monitored monthly during 2025 using quantitative PCR-based absolute quantification. Roots, one-year-old shoots and annual shoots were sampled monthly from 21 infected trees of Prunus armeniaca, Prunus domestica, Prunus persica and Prunus salicina. Phytoplasma titer varied with plant part and sampling month, and differences were also observed among the examined host groups. Because cultivar and rootstock were linked to species, these differences cannot be attributed to species alone. Roots provided the most consistent year-round detection, whereas above-ground tissues showed stronger monthly variation during the studied period. P. domestica displayed the most distinct pattern: roots remained frequently positive, while one-year-old shoots and particularly annual shoots had lower detection rates and lower phytoplasma titers. Multilocus genotyping detected ten multilocus profiles, but their uneven distribution among only 21 trees precluded a reliable evaluation of their relationship with phytoplasma titer or symptom expression.

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. [Formula: see text] Copyright © 2026 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

Abstract Syndrome Basses Richesses (SBR) is a phloem-limited disease of sugar beet caused by ‘ Candidatus Arsenophonus phytopathogenicus’ (ARSEPH), a bacterium currently considered a facultative endosymbiont of its insect vector, Pentastiridius leporinus . Due to increasing disease incidence and the need for reliable vector-free resistance screening methods, this study evaluated alternative ARSEPH transmission routes using chip grafting and the parasitic plant dodder ( Cuscuta campestris ). Given that Arsenophonus species are unculturable and their insect vectors have complex life cycles so laboratory propagation is labor intensive, the development of vector-independent systems is desirable for controlled pathogen transmission and host–pathogen interaction studies. Chip grafting enabled successful ARSEPH transmission to sugar beet, though with low efficiency (~ 9.6%) and delayed pathogen detection. While C. campestris acquired and maintained high ARSEPH titers, it failed to transmit the bacterium to healthy plants, indicating limited systemic movement. In contrast, vector-mediated transmission via P. leporinus achieved 100% efficiency within 25 days, confirming a strong biological compatibility. These findings underscore the limitations of non-vector-based approaches for phloem-restricted pathogens and highlight the need for methodological optimization. The study provides key insights into ARSEPH’s complex transmission biology and supports a functionally specialized association with its insect vector.

During field surveys conducted in 2024 and 2025 in pepper production areas in Altunhisar distinct of Niğde province, Türkiye, plants showing phytoplasma-like symptoms were observed. A total of 25 symptomatic pepper samples were collected, and total nucleic acids were extracted using the CTAB method. The samples were analyzed for phytoplasma infection by PCR amplification of the 16S rRNA gene region. In addition, two asymptomatic pepper plants were included as negative controls. PCR analysis revealed that all samples showing symptoms produced amplicons of the expected size, while no amplification was obtained from the asymptomatic plants. Sequencing and phylogenetic analyses revealed that the detected phytoplasma belonged to the 16SrVI-A subgroup, corresponding to ‘Candidatus Phytoplasma trifolii’. The subgroup assignment was further confirmed by in silico (virtual) RFLP analysis of the 16S rRNA gene. The representative 16S rRNA gene sequence of one strain obtained in this study was deposited in the GenBank database (GenBank accession number PX885052). Based on the available literature, this study provides the first molecular evidence of ‘Candidatus Phytoplasma trifolii’ infection in pepper plants grown in Niğde province, Türkiye. The findings contribute to the current knowledge on the distribution of pepper-infecting phytoplasmas in Türkiye and emphasize the need for further epidemiological and vector-related studies.

Abstract Phytoplasma diseases are threat to sesame and its wild relatives, but information on their occurrence in exotic Sesamum species is limited. During field evaluation, Sesamum angolense , S. angustifolium , and S. latifolium exhibited characteristic phyllody and witches' broom symptoms, including virescence, leafy floral organs, polysepalous calyx, polypetalous corolla, flattened stamens, shortened internodes, excessive shoot proliferation, and leaf twisting and curling. Molecular detection using universal phytoplasma primers P1/P7 and tuf gene-specific primers fTufAy/rTufAy amplified products of approximately 1.8 kb and 940 bp, respectively, from symptomatic plants. Sequence analysis of the 16S rRNA and tuf genes revealed 99–100% nucleotide identity with members of the 'Candidatus Phytoplasma asteris ' group. Phylogenetic reconstruction based on both loci consistently assigned the isolates to the 16SrI-B subgroup of 'Ca. Phytoplasma asteris'. To the best of our knowledge, this is the first report of phytoplasma infection in S. angolense , S. angustifolium , and S. latifolium , characterized at the strain level using both 16S rRNA and tuf gene sequences. The findings expand the known host range of 16SrI-B phytoplasmas and demonstrate that these wild Sesamum species, which are valuable components of the tertiary gene pool of cultivated sesame, can serve as natural reservoirs of phytoplasma inoculum.

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 Huanglongbing (HLB), primarily induced by the phloem-restricted bacterium Candidatus Liberibacter asiaticus (CLas), poses a significant threaten to citrus production worldwide. CLas deploys Sec-dependent effectors (SDEs) to manipulate host cellular processes, yet the underlying virulence mechanisms remain incompletely understood. Here, we identify SDE4580, a conserved CLas effector localized to the plant plasma membrane, as a key virulence factor that promotes pathogen proliferation and suppresses host immunity across citrus and Arabidopsis. Transcriptomic analysis reveals that SDE4580 reprograms membrane-associated biological processes and defense responses. Through yeast two-hybrid screening and biochemical validation, we demonstrate that SDE4580 physically interacts with the citrus vesicle trafficking regulator CsPRA1.E, a Prenylated Rab Acceptor 1 family protein. Functional assays establish CsPRA1.E as a positive regulator of immunity that promotes apoplastic secretion of defense-related proteins and enhances resistance to Xanthomonas citri subsp. citri. Crucially, SDE4580 antagonizes CsPRA1.E-mediated resistance by suppressing defense protein secretion without inducing target degradation. These findings uncover a novel pathogenic strategy whereby CLas subverts host endomembrane trafficking to disarm plant immunity. Targeting the SDE4580-CsPRA1.E interface may offer a promising avenue for developing durable HLB control strategies.

‘Candidatus Liberibacter solanacearum’ (Lso) haplotype D, transmitted by the carrot psyllid Bactericera trigonica, is a major threat to carrot production, particularly in the Mediterranean Basin and the Middle East. We investigated the impact of plant age at the time of inoculation on symptom development and yield under greenhouse and outdoor conditions. In greenhouse trials, 1- and 2-month-old plants were inoculated with psyllids harboring Lso. Both age groups showed reduced taproot weight compared with controls, but younger plants were more severely affected, exhibiting 90% yield reduction, while 2-month-old plants showed a 30% reduction. Witches’ broom symptoms appeared earlier in plants inoculated at 1 month (5 weeks postinoculation); however, within 2 weeks, plants inoculated at 2 months reached comparable levels of symptom incidence and severity. Additional greenhouse experiments using Lso-free psyllids confirmed that yield losses and disease symptoms were specifically due to Lso infection and not psyllid feeding. In outdoor trials, we tested the effects of inoculation at 7, 11, 15, and 19 weeks postsowing (wps) on disease incidence and yield. Significant yield losses occurred only in plants inoculated at 7 and 11 wps, with up to 45% reduction by harvest time. Symptoms began to appear at 20 wps in plants inoculated at 7 and 11 wps. At 23 wps, symptoms also appeared in plants inoculated at 15 wps, with a similar incidence to that observed in the earlier inoculation groups. Molecular detection of Lso at harvest (23 wps) confirmed that all plants inoculated at 19 wps or earlier were infected. These results demonstrate that carrots are highly susceptible to Lso-induced yield losses at the earlier stages of growth and highlight the importance of managing early season vector pressure to minimize economic damage.