Publications

4560

Abstract‘Candidatus Phytoplasma (Ca. P.) solani’ is associated with Bois noir (BN) of grapevine and stolbur of solanaceous plants and is primarily transmitted by Hyalesthes obsoletus Signoret. Four tuf‐a and five tuf‐b1 ‘Ca. P. solani’ strains were transmitted to tomato plants (cv. Micro‐Tom) to set the basis for studying molecular interactions between different strains of the pathogen and host plants. The strains were acquired by using bait‐plants and by capturing H. obsoletus adults on bindweed and stinging nettle in vineyards of Friuli Venezia Giulia (northeastern Italy) with a high prevalence of BN. Captured insects were forced to feed on healthy tomato plants to induce infection. All strains obtained from symptomatic plants and confirmed by real‐time PCR were maintained on tomato through grafting. Successively, the strains were characterised by macroscopic and microscopic symptoms induced in the host, Multi‐Locus Sequence Typing (MLST) based on tuf, secY, stamp, and vmp1 genes, in‐planta spread and multiplication patterns. Molecular typing distinguished the strains into five lineages comprised in three clusters: one including strains of tuf‐a genotype and two including strains of tuf‐b1 genotype. Quite different symptoms were induced on tomatoes by strains belonging to the two tuf genotypes; infection by tuf‐a strains resulted in plant decline around 95–100 days after grafting and absence of cauliflower‐like inflorescence with symptoms of phyllody and virescence, which were usually associated with tuf‐b1 strains. The different symptoms, the outcome of disease, and the ultrastructural observation performed on sieve elements suggested a higher virulence of tuf‐a strains in tomato. Overall, our results propose that genomic variability of ‘Ca. P. solani’ strains should be extensively explored to determine possible associations with type of symptoms and strain virulence.

ABSTRACT Huanglongbing (HLB), caused by Candidatus Liberibacter asiaticus (CLas), is the most devastating citrus disease worldwide. Developing effective therapies remains a major challenge, as CLas cannot be cultured in vitro and colonizes the host phloem systemically. This study presents a rapid, reproducible, and cost-effective in vivo platform for screening bacteriostatic and bactericidal compounds using CLas-infected citron (Citrus medica (L.) Osbeck) stem cuttings. Among seven citrus genotypes tested, citron stem cuttings exhibited superior rooting performance and uniform vegetative growth. Four propagation protocols were developed and assessed based on the dynamics of rooting and shoot growth, CLas colonization, and the response to oxytetracycline (OTC) treatment. CLas+ stem cuttings were treated with OTC via drenching at different developmental root stages. The roots or new vegetative flushes were sampled for bacterial quantification by qPCR. In Protocol 2, in which treatments were applied and sampled 14 and 35 days after planting (DAP) respectively, OTC-treated roots achieved the highest suppression of CLas and the lower incidence of CLas+ rooted cuttings compared to non-treated roots. Time-course analysis showed that OTC delayed bacterial establishment in root tissues, with maximal suppression observed at 35 DAP. The proposed protocols simulate the natural progression of systemic infection in citrus plants, allow the assessment of phytotoxicity, and offer a scalable technology that does not underestimate the efficacy of future bactericidal candidates. This platform significantly reduces time and cost compared to traditional seedling or nursery tree experiments and enhances the early-phase screening of antimicrobial compounds. Altogether, this stem cutting-based approach represents a biologically relevant, scalable tool to accelerate therapeutic discovery and strengthen integrated HLB management strategies.

Abstract Background Candidatus Phytoplasma infection is among the most destructive plant diseases, characterized by phyllody, witches broom, virescence etc. Sesame (Sesamum indicum L.), one of the oldest cultivated oilseed crops, valued for its nutritional and medicinal properties, is highly susceptible to phytoplasma infection, causing substantial annual yield losses. The present study aimed to investigate the metabolomic and molecular alterations in sesame plants as a consequence of phytoplasma infection in two distinct tissue types, leaves and flowers, using a multi-omics approach. Results The study revealed that phytoplasma infection significantly alters the expression of floral homeobox and meristem identity genes in an antagonistic manner, resulting in the development of leaf-like traits in floral tissues. Integrated analyses of transcriptomic and metabolomic datasets showed that control leaves, control flowers, infected leaves, and infected flowers each displayed distinct metabolomic and transcriptomic profiles. Metabolomic profiling demonstrated major changes in pathways such as porphyrin metabolism, brassinosteroid metabolism, and phenylpropanoid biosynthesis. Complementary KEGG pathway enrichment analysis of transcriptome data confirmed strong enrichment of secondary metabolite biosynthesis in both tissue types upon infection. Tissue-specific responses were evident. Floral tissues accumulated green pigments due to increased porphyrin biosynthesis and reduced degradation, while leaves showed simultaneous upregulation of both biosynthesis and breakdown pathways of porphyrins. Floral tissues exhibited stronger stress-associated responses, including upregulation of genes related to stress enzymes, phenylpropanoids, and lignification-related metabolites. In contrast, certain compounds such as lignans were specifically accumulated in leaves upon infection. These observations were further supported by biochemical, histological, and qRT-PCR assays. Conclusion This study provides the first clear evidence of tissue-specific metabolic reprogramming in sesame under Ca. Phytoplasma infection through integrated transcriptomic and metabolomic analyses. These findings improve our understanding of host-pathogen interactions and offer a basis for strategies to reduce phytoplasma-induced yield losses.

‘Candidatus Liberibacter asiaticus’ (CLas), the agent associated with the Huanglongbing citrus disease, is commonly detected using quantitative PCR (qPCR) with hydrolysis probes. Internal standards are typically included in the qPCR assays to reduce the risk of false negatives caused by inhibitors. When the internal standard is detected but CLas is not, it is generally assumed that the pathogen is absent from the tested sample. However, our study shows that trace amounts of CLas may go undetected if the internal standard is either overly abundant or too dissimilar to CLas. To overcome these limitations, we developed a synthetic internal standard (IS) that uses the same primer as the CLas target sequence, along with three to four downstream nucleotides, but with a unique internal sequence derived from smooth hammerhead shark (Sphyrna zygaena; IS-SHK). This sequence matches the guanine-cytosine content and melting temperature of the CLas target and was specifically selected to minimize potential interference from other nucleic acid materials in citrus samples. To minimize competition between the IS-SHK standard and the CLas target, an average of only 21 molecules of IS-SHK standard is added to each qPCR reaction. Therefore, when IS-SHK standard is detected at expected levels and CLas is not, the absence of CLas is confirmed rather than inhibition of the detection. Conversely, the absence of both IS-SHK standard and CLas suggests the presence of a qPCR inhibitor, warranting retesting of the sample.

Abstract Citrus Huanglongbing (HLB) is the most destructive disease in citriculture, mainly caused by Candidatus Liberibacter asiaticus (CLas). However, the immune response of citrus to CLas at the cellular level remains to be elucidated. In this study, the first single-cell atlas of rough lemon (Citrus jambhiri Lush.) root apexes were generated using single-nucleus RNA sequencing at 20 weeks post-inoculation with CLas. According to gene expression patterns, the single-cell atlas was partitioned into 20 transcriptionally distinct clusters, and five cell types were identified within these clusters. A significant number of defense-related genes were co-upregulated across the five cell types following CLas infection, whereas genes involved in signal transduction pathways, such as tubulin beta-6 chain (TUBB1) and the phospholipase D alpha 1 (PLD1), were concurrently downregulated. Based on pseudotime trajectory analysis, the key pathways and genes involved in the coordination of cell differentiation and resistance in citrus under CLas infection were characterized. Following CLas infection, the development of phloem cells was significantly delayed, and the differentiation of cambium cells into xylem cells was evident. The expression of genes associated with lignin synthesis was significantly upregulated in these cells. The reduction in phloem cell differentiation and the enhanced differentiation of cambium cells into defense-related xylem cells may represent the primary vascular immune mechanisms exhibited by citrus plants in response to CLas infection. Additionally, DNA-binding one zinc finger transcription factor DOF2.4 was found to potentially serve dual roles in regulating vascular cell development and inducing plant resistance against CLas. In conclusion, this study collectively provides insights into the cellular innate immunity responses of citrus to CLas infection. These findings hold significant implications for the sustainable development of citriculture amidst the ongoing global HLB epidemic, and offer novel insights into vascular immunity and plant defense responses.