AbstractCassava witches’ broom CWB disease is associated with the presence of phytoplasmas. Typical symptoms include witches’ broom, shoot proliferation, short internodes and stunting. Severely infected plants decline, resulting in yield losses of up to 80%. CWB has been reported in several cassava growing regions in Thailand. In total, 25 CWB symptomatic cassava plant samples were collected from 9 plots in Kanchanaburi Province in November 2023. Detection of CWB phytoplasma was carried out using nested PCR assay to amplify the 16S rRNA gene using primers P1/P7, followed by R16F2n/R16R2. A target DNA fragment of approximately 1,800 bp was obtained by PCR in 15 samples, and a 1,200 bp fragment from nested PCR was obtained in 24 samples. Sample KRI-M-4 (GenBank accession number PQ333137) was used for molecular identification. It showed 98.65% identity with the reference strain ‘Candidatus Phytoplasma solani’ (GenBank accession number AF248959). BLAST analysis in the GenBank, NCBI database showed 99.07% similarity to a ‘Ca. P. solani’ strain from Japan (GenBank accession number LC460259). The partial stamp gene of KRI-M-4 strain was cloned, sequenced, and analyzed in the GenBank, NCBI database showing 100% identity to a ‘Ca. P. solani’ strain from Thailand (GenBank accession number MW464308). Phylogenetic tree analysis and a heatmap of pairwise distance analysis of the 16S rDNA sequences and partial stamp gene sequences confirmed that the KRI-M-4 strain clustered with phytoplasmas in the 16SrXII group. This is the first report of the presence of a ‘Ca. P. solani’ strain associated with CWB disease, in plants exhibiting witches’ broom, yellow leaf, and short internode symptoms in Thailand.
AbstractGrapevine yellows (GY) is a destructive phytoplasma disease complex in viticultural regions worldwide, mainly associated with the presence of ‘Candidatus Phytoplasma solani’ (“bois noir”, 16SrXII-A) and 16SrV group phytoplasmas (“flavescence doree”). Field surveys in 2021-2022 in Hatay and Kahramanmaras (Türkiye) investigated phytoplasma occurrence in grapevines, weeds, and insect vectors. Typical GY symptoms were observed in grapevine and bindweed (Convolvulus arvensis). ‘Ca. P. solani’ was detected in one grapevine and one bindweed plants among the 25 grapevine and 19 bindweed samples tested by PCR and sequencing analysis. Among the 3,086 leafhoppers (28 species), 12 individuals from six species were positive for phytoplasmas, with the highest infection rates in Psammotettix provincialis, Empoasca sp. and Balclutha hebe. Sequencing confirmed ‘Ca. P. solani’ presence in three insect species, grapevine, and bindweed, while ‘Ca. P. mali’ (apple proliferation phytoplasma, 16SrX-A) was detected in six leafhopper individuals. These results provide the first molecular evidence of ‘Ca. P. solani’ and ‘Ca. P. mali’ in leafhoppers collected from vineyards in Türkiye and highlight the possible epidemiological role of diverse leafhopper species in GY spread.
AbstractBesides viral diseases, phytoplasma-associated diseases pose a significant threat to okra cultivation in India. During 2022-2025 seasons, okra plants exhibiting characteristic phytoplasma symptoms such as little leaf, shoot proliferation, bud proliferation and bunchy crown were observed in Kerala, New Delhi and Tripura states of India. Molecular detection using phytoplasma-specific primers P1/Tint and R16F2n/R16R2 amplified DNA fragments of about 1.25 kb of the 16S rRNA gene from symptomatic okra leaf samples, while asymptomatic samples showed no amplifications. Pairwise sequence analysis of the 16S rRNA gene revealed almost 100% identity among the okra phytoplasma strains in the study and ‘Candidatus Phytoplasma asteris’ strains. Phylogenetic analysis further clustered these okra phytoplasma strains with ‘Ca. P. asteris’ strains. In silico RFLP analysis employing 17 restriction endonucleases confirmed the affiliation of the okra phytoplasma strains within the 16SrI-B subgroup, with a similarity coefficient of 1.00. Although the ‘Ca. P. asteris’ has been reported earlier in India, the present study highlights the prevalence and importance of ‘Ca. P. asteris’ strains occurrence in okra crops across three Indian states, underscoring their epidemiological importance.
AbstractWeligama coconut leaf wilt disease (WCLWD) is one of the devastating diseases in the Southern province of Sri Lanka, causing drastic yield reductions in coconut. The agent associated with WCLWD is a phytoplasma, which is an intracellular bacterium. Accurate molecular detection of phytoplasmas is important for effective disease management, especially due to the long latent period, during which the disease could be spread from non-symptomatic coconut palms. Two sampling rounds were conducted using 30 selected WCLWD palms, with 10 each from mild, moderate and severe disease categories and 10 asymptomatic coconut palms as control. DNA was extracted from the midrib of milky white emerging bud leaf tissues using a CTAB method, and nested PCR was performed to detect phytoplasmas. Extracted DNA was purified using Qiagen DNA purification kit to evaluate the effect of DNA purification on PCR detection. PCR results were assessed and compared between pre-purified and purified DNA samples. The highest PCR positivity was observed in the moderate disease severity stage after the DNA purification. However, upon DNA purification PCR positivity increased to 30%, 60% and 30% respectively for mild, moderate and severe palm categories. None of the asymptomatic control samples showed PCR positivity. In conclusion, DNA purification improved the quality of extracted DNA and efficiency of PCR detection, and the moderate disease severity stage was identified to be the best stage for detection of the WCLWD phytoplasma. The findings would be useful in WCLWD management programs.
AbstractGeorgia is one of the primary centers of Vitis vinifera domestication and maintains a rich and diverse grapevine germplasm comprising over 500 autochthonous cultivars. Recent investigations have revealed distinct genetic identity and potential tolerance to major grapevine pathogens of this gene pool. Among these, grapevine yellows (GY) diseases, principally “flavescence dorée” and “bois noir” (BN), represent severe threats to viticulture worldwide. Surveys conducted in Georgian vineyards documented for the first time the presence of ‘Candidatus Phytoplasma solani’ (BN-associated phytoplasma) in both grapevines and Convolvulus arvensis, whereas the “flavescence dorée” phytoplasma was not detected. Molecular characterization revealed high genetic diversity among ‘Ca. P. solani’ strains, mainly linked to the bindweed-related pathosystem, and indicated that Georgian grapevine varieties display lower susceptibility to BN compared to international cultivars, which exhibited severe symptoms and yield loss. Subsequent large-scale surveys identified phytoplasmas belonging to the 16SrV group in Georgian vineyards, likely introduced through imported propagation material. Complementary field trials in Piedmont (northwestern Italy), a region heavily infected by “flavescence dorée”, demonstrated that several Georgian cultivars infected by the “flavescence dorée” phytoplasma (genotype M54) exhibited mild or no symptoms and preserved stable berry and wine quality traits, indicating tolerance to the infection. The unique genetic diversity and reduced susceptibility of Georgian V. vinifera varieties to GY make this germplasm a valuable genetic resource for breeding programs aimed at enhancing phytoplasma tolerance while maintaining high oenological quality. The implementation of mandatory certification of grapevine planting materials produced in Georgia (since 2024) and the application of sensitive molecular diagnostic tools will be crucial to prevent phytoplasma spread and ensure the production of healthy nursery stock.
AbstractMaize bushy stunt phytoplasma is a pathogenic bacterium transmitted by the corn leafhopper, Dalbulus maidis (Hemiptera: Cicadellidae), in a persistent-propagative manner. This phytoplasma is a key component of a reemerging disease complex that poses a major threat to maize production across the American continent. While some studies have reported genetic variation among maize bushy stunt phytoplasma strains, it remains unclear whether these variations influence the transmission efficiency by D. maidis. Here, it was assessed the efficiency of acquisition by its insect vector. Six Brazilian strains were obtained from field samples and maintained in greenhouse through serial transfer using D. maidis from a healthy colony. Acquisition efficiency was evaluated by exposing groups of 100 third-instar D. maidis nymphs to maize bushy stunt phytoplasma-infected plants. The findings indicate that these Brazilian strains exhibit differential acquisition efficiencies. A higher likelihood of acquisition may be linked to host manipulation strategies that enhance pathogen persistence and dissemination to new niches.
AbstractPhytoplasmas are wall-less and pleomorphic bacterial pathogens that cause considerable yield losses in more than 1,000 plant species worldwide. They are associated with the presence of different symptoms including witches’ broom, phyllody, virescence, yellowing. The aim of the present work was to analyze the changes in protein contents, phenylalanine ammonia-lyase (PAL), peroxidase (POX), catalase (CAT), polyphenol oxidase (PPO) in field-grown chickpea plants (Cicer arietinum) in the presence of phytoplasma infection. The activities of PAL, PPO and POX were observed to increase in the infected plants compared with healthy plants. The amount of total soluble proteins, chlorophyll content and catalase activity was significantly higher in healthy leaves. In conclusion the observed alterations could be considered as the response and the adaptation of chickpea plants to the presence of phytoplasmas.
AbstractDuring field surveys conducted in 2024, Rudbeckia species plants (Asteraceae) exhibiting symptoms of phyllody, virescence, axillary shoot proliferation, and stunting were observed in public gardens in Istanbul, Türkiye. To investigate possible phytoplasma presence, symptomatic plants were analyzed by polymerase chain reaction (PCR) using universal primers targeting ribosomal 16S rRNA gene. All symptomatic samples tested positive and PCR with primer pair P1/P7 followed by nested PCR with R16F2n/R16R2, consistently yielded amplicons of the expected size (1.25 kb). Sanger sequencing of the 16S rRNA gene and BLASTn analysis against the NCBI database revealed 99.30–99.44% sequence identity with ‘Candidatus Phytoplasma asteris’ and other members of the aster yellows group (16SrI). Phylogenetic analysis further confirmed that the sequences clustered within phytoplasma strains enclosed in subgroup 16SrI-B. This study provides the first evidence of phytoplasma infection in Rudbeckia species in Türkiye and expands the documented host range of phytoplasmas in this region of Türkiye.
AbstractCitrus stubborn disease (CSD) was originally discovered in California in 1915 and it has been one of the most important problems which affects the citrus industry in the East Mediterranean region of Türkiye since 1950’s. The first studies began in 1965 and were limited to survey and biological indexing until 1980s. Spiroplasma citri as the causal agent of stubborn disease was cultured for the first time from symptomatic citrus plants and other hosts in 1987 in Türkiye. Then polyclonal antisera was produced against local S. citri isolates, one dimensional gel-electrophoresis was used for further characterization and spiroplasmas were observed under scanning and transmission electron microscopes both in infected citrus tissues and also in culture. S. citri was also isolated from the Catharanthus roseus placed in citrus orchards where stubborn disease was detected and Exitianus capicola, collected from the same plots indicating the first evidence of natural possible insect transmission of stubborn disease. In the following years the epidemiological aspects of CSD were investigated in more details and new leafhopper and herbaceous hosts of S. citri were reported. Because of CSD diagnosis is difficult due to low and variable concentrations of S. citri in diseased trees and the random distribution of the pathogen, recently molecular detection techniques like PCR has been used both for diagnostic and genetic diversity studies of local S. citri isolates. The future studies will be focued on functional genomics in host-pathogen interaction using transcriptomic and proteomic approaches to learn more about the mechanisms of the S. citri pathogenicity to develop new diagnostic methods and implement plant protection strategies to control citrus stubborn disease.
AbstractSince 2021, symptoms of little leaf disease have been observed in periwinkle plants grown in Eram garden, Fars province, Iran. The agent of the Eram garden periwinkle little leaf (EGPLL) disease was transmitted to periwinkle and eggplant plants through dodder and induced phytoplasma type symptoms. Phylogenetic and iPhyClassifier analyses using a 1250 bp fragment of the 16S rRNA gene, obtained via nested PCR, cloning and sequencing identified the EGPLL phytoplasma as a ‘Candidatus Phytoplasma asteris’ strain belonging to the 16Sr group I, subgroup F. This is the first report of a 16SrI-F phytoplasma strain naturally occurring in periwinkle in Iran.
AbstractBermudagrass (Cynodon dactylon) is an invasive weed severely infected by Bermudagrass white leaf (BGWL) phytoplasma disease worldwide. During a survey in April 2025, Bermudagrass samples exhibiting white leaf symptoms were collected from Madhya Pradesh, Central India. The total DNA was extracted from symptomatic and asymptomatic leaf samples by a CTAB method and nested PCR assay was performed using universal primers for phytoplasma detection targeting 16S rRNA gene. Resulting amplicons (about 1.2 kb) were purified, sequenced and submitted to GenBank under accession numbers PX442054-PX442057. The sequence analysis revealed about 99% sequence identities and close phylogenetic relationships with ‘Candidatus Phytoplasma cynodontis’ (16SrXIV-A subgroup). The iPhyClassifier RFLP analysis of the 16S rRNA gene sequence indicated that these strains had a similarity coefficient of 0.85 to 0.89 to the reference strain of the phytoplasmas in the subgroup 16SrXIV-A suggesting that it may be a new lineage of ‘Ca. P. cynodontis’ which needs further confirmation.
AbstractPhytoplasmas represent a group of plant pathogens implicated in numerous diseases affecting a wide range of wild and cultivated plant species across the globe. They are prokaryotes that lack cell wall, and are localized in plant tissues with active phloem, such as shoots, midribs, and petioles of young leaves, making these organs ideal for sampling. Rapid and accurate phytoplasma detection, together with understanding their relationship with host plants, is crucial for managing phytoplasma-associated diseases. Most scientific articles emphasize the importance of rapidly processing phytoplasma-infected plant samples to avoid the damaging effects of oxidation. However, when dealing with a large number of samples and biological replicates, processing samples within a reasonable period is almost impossible. This motivated to conduct experiments on long-term samples storage and faster processing of large sample numbers. Additional difficulties were encountered during the sample processing, particularly when homogenizing the phloem of woody plants. Producing high-quality DNA is crucial for molecular studies, as is facilitating the processing of numerous samples. By combining a specific storage method for samples and using a dedicated grinding machine, it was possible to efficiently obtain phytoplasma DNA with a high quality and extend the seasonal work throughout the year.
