Witches’-broom (WB, excessive initiation, and outgrowth of axillary buds) is one of the remarkable symptoms in plants caused by phytoplasmas, minute wall-less intracellular bacteria. In healthy plants, axillary bud initiation and outgrowth are regulated by an intricate interplay of nutrients (such as sugars), hormones, and environmental factors. However, how these factors are involved in the induction of WB by phytoplasma is poorly understood. We postulated that the WB symptom is a manifestation of the pathologically induced redistribution of sugar and phytohormones. Employing potato purple top phytoplasma and its alternative host tomato (Solanum lycopersicum), sugar metabolism and transportation, and the spatiotemporal distribution of phytohormones were investigated. A transmission electron microscopy (TEM) analysis revealed that starch breakdown was inhibited, resulting in the degradation of damaged chloroplasts, and in turn, premature leaf senescence. In the infected source leaves, two marker genes encoding asparagine synthetase (Sl-ASN) and trehalose-6-phosphate synthase (Sl-TPS) that induce early leaf senescence were significantly up-regulated. However, the key gibberellin biosynthesis gene that encodes ent-kaurene synthase (Sl-KS) was suppressed. The assessment of sugar content in various infected tissues (mature leaves, stems, roots, and leaf axils) indicated that sucrose transportation through phloem was impeded, leading to sucrose reallocation into the leaf axils. Excessive callose deposition and the resulting reduction in sieve pore size revealed by aniline blue staining and TEM provided additional evidence to support impaired sugar transport. In addition, a spatiotemporal distribution study of cytokinin and auxin using reporter lines detected a cytokinin signal in leaf axils where the axillary buds initiated. However, the auxin responsive signal was rarely present in such leaf axils, but at the tips of the newly elongated buds. These results suggested that redistributed sucrose as well as cytokinin in leaf axils triggered the axillary bud initiation, and auxin played a role in the bud elongation. The expression profiles of genes encoding squamosa promoter-binding proteins (Sl-SBP1), and BRANCHED1 (Sl-BRC1a and Sl-BRC1b) that control axillary bud release, as determined by quantitative reverse transcription (qRT)-PCR, indicated their roles in WB induction. However, their interactions with sugars and cytokinins require further study. Our findings provide a comprehensive insight into the mechanisms by which phytoplasmas induce WB along with leaf chlorosis, little leaf, and stunted growth.

Phytoplasmas are small, intracellular bacteria that infect a vast range of plant species, causing significant economic losses and impacting agriculture and farmers’ livelihoods. Early and rapid diagnosis of phytoplasma infections is crucial for preventing the spread of these diseases, particularly through early symptom recognition in the field by farmers and growers. A symptom database for phytoplasma infections can assist in recognizing the symptoms and enhance early detection and management. In this study, nearly 35,000 phytoplasma sequence entries were retrieved from the NCBI nucleotide database using the keyword “phytoplasma” and information on phytoplasma disease-associated plant hosts and symptoms was gathered. A total of 945 plant species were identified to be associated with phytoplasma infections. Subsequently, links to symptomatic images of these known susceptible plant species were manually curated, and the Phytoplasma Disease Symptom Database (iPhyDSDB) was established and implemented on a web-based interface using the MySQL Server and PHP programming language. One of the key features of iPhyDSDB is the curated collection of links to symptomatic images representing various phytoplasma-infected plant species, allowing users to easily access the original source of the collected images and detailed disease information. Furthermore, images and descriptive definitions of typical symptoms induced by phytoplasmas were included in iPhyDSDB. The newly developed database and web interface, equipped with advanced search functionality, will help farmers, growers, researchers, and educators to efficiently query the database based on specific categories such as plant host and symptom type. This resource will aid the users in comparing, identifying, and diagnosing phytoplasma-related diseases, enhancing the understanding and management of these infections.

Phytoplasmas are small phloem-restricted and insect-transmissible bacteria that infect many plant species, including important crops and ornamental plants, causing severe economic losses. Our previous studies screened phytoplasmas in hundreds of leafhoppers collected from natural habitats worldwide and identified multiple genetically different phytoplasmas in seven leafhopper species (potential insect vectors). As an initial step toward determining the impact of these phytoplasmas on the ecosystem, ribulose 1,5-biphosphate carboxylase large subunit ( rbcL), a commonly used plant DNA barcoding marker, was employed to identify the plant species that the phytoplasma-harboring leafhoppers feed on. The DNA of 17 individual leafhoppers was PCR amplified using universal rbcL primers. PCR products were cloned, and five clones per amplicon were randomly chosen for Sanger sequencing. Moreover, Illumina high-throughput sequencing on selected PCR products was conducted and confirmed no missing targets in Sanger sequencing. The nucleotide BLAST results revealed 14 plant species, including six well-known plant hosts of phytoplasmas such as tomato, alfalfa, and maize. The remaining species have not been documented as phytoplasma hosts, expanding our knowledge of potential plant hosts. Notably, the DNA of tomato and maize (apparently cultivated in well-managed croplands) was detected in some phytoplasma-harboring leafhopper species sampled in non-crop lands, suggesting the spillover/spillback risk of phytoplasma strains between crop and non-crop areas. Furthermore, our results indicate that barcoding (or metabarcoding) is a valuable tool to study the three-way interactions among phytoplasmas, plant hosts, and vectors. The findings contribute to a better understanding of phytoplasma host range, host shift, and disease epidemiology.

Phytoplasmas are intracellular plant pathogens that heavily rely on host cell nutrients for survival and propagation due to their limited ability to synthesize essential substrates. The endoplasmic reticulum (ER), which plays a vital role in various cellular processes, including lipid and protein biosynthesis, is an attractive target for numerous intracellular pathogens to exploit. This study investigated the impact of potato purple top (PPT) phytoplasma infection on the ER in tomato plants. Abnormal accumulation of ER-resident proteins, disrupted ER network structures, and formation of protein aggregates in the phloem were observed using confocal microscopy and transmission electron microscopy, indicating a phytoplasma-infection-induced disturbance in ER homeostasis. The colocalization of phytoplasmas with the accumulated ER-resident proteins suggests an association between ER stress, unfolded protein response (UPR) induction, and phytoplasma infection and colonization, with the ER stress response likely contributing to the host plant’s defense mechanisms. Quantitative real-time PCR revealed a negative correlation between ER stress/UPR activation and PPT phytoplasma titer, implying the involvement of UPR in curbing phytoplasma proliferation. Inducing ER stress and activating the UPR pathway effectively decreased phytoplasma titer, while suppressing the ER-resident protein, binding immunoglobulin protein (BiP) increased phytoplasma titer. These results highlight the ER as an intracellular battleground where phytoplasmas exploit host components for survival and multiplication, while host plants deploy defense mechanisms to counteract the invasion. Understanding the intricate interactions between phytoplasmas and plant hosts at the subcellular level, particularly within the ER, provides valuable insights for developing new strategies to control phytoplasma diseases.

Phytoplasmas are intracellular pathogenic bacteria that infect a wide range of plant species, including agriculturally important crops and ornamental trees. However, our understanding of the relationship between symptom severity, disease progression, and phytoplasma concentration remains limited due to the inability to inoculate phytoplasmas mechanically into new plant hosts. The present study investigated phytoplasma titer dynamics and symptom development in periwinkle and tomato, both infected with the same potato purple top (PPT) phytoplasma strain using a small seedling grafting approach. Virescence, phyllody, and witches’-broom (WB) symptoms sequentially developed in periwinkle, while in tomato plants, big bud (BB, a form of phyllody), cauliflower-like inflorescence (CLI), and WB appeared in order. Results from quantitative polymerase chain reaction (qPCR) targeting the PPT phytoplasma’s 16S rRNA gene revealed that in both host species, phytoplasma titers differed significantly at different infection stages. Notably, the highest phytoplasma concentration in periwinkles was observed in samples displaying phyllody symptoms, whereas in tomatoes, the titer peaked at the BB stage. Western blot analysis, utilizing an antibody specific to PPT phytoplasma, confirmed substantial phytoplasma presence in samples displaying phyllody and BB symptoms, consistent with the qPCR results. These findings challenge the conventional understanding that phytoplasma infection dynamics result in a higher titer at later stages, such as WB (excessive vegetative growth), rather than in the early stage, such as phyllody (abnormal reproductive growth). Furthermore, the PPT phytoplasma titer was markedly higher in periwinkles than in tomato plants, indicating differing susceptibilities between the hosts. This study reveals distinct host responses to PPT phytoplasma infection, providing valuable insights into phytoplasma titer dynamics and symptom development, with implications for the future management of agricultural disease.

Nigerian papaya bunchy top (NGPBT) phytoplasma was first identified in diseased papaya plants growing in Ibadan, Oyo State, Nigeria (Kazeem et al. 2021). The NGPBT phytoplasma is a ‘Candidatus Phyt...