Phytoplasma Infection Research Articles

In situ Detection and Investigation of the Ultrastructural Modiflcations of Phytoplasma Infection on Daffodil (Narcissus tazetta) Using a Combined Microscopy Approaches

In situ Detection and Investigation of the Ultrastructural Modiflcations of Phytoplasma Infection on Daffodil (Narcissus tazetta) Using a Combined Microscopy Approaches

Flavescence Dorée Strain-Specific Impact on Phenolic Metabolism Dynamics in Grapevine (Vitis vinifera) throughout the Development of Phytoplasma Infection.

Flavescence dorée phytoplasma (FDp) is a phytopathogenic bacterium associated with Grapevine yellowS disease, which causes heavy damage to viticultural production. Epidemiological data revealed that some FDp strains appear to be more widespread and aggressive. However, there is no data on mechanisms underlying the variable pathogenicity among strains. In this research, we employed chromatographic and spectrophotometric techniques to assess how two strains of FDp influence the levels of grapevine phenolic compounds, which are frequently utilized as indicative markers of stress conditions. The results pointed to the upregulation of all branches of phenolic metabolism through the development of infection, correlating with the increase in antioxidative capacity. The more aggressive strain M54 induced stronger downregulation of phenolics' accumulation at the beginning and higher upregulation by the end of the season than the less aggressive M38 strain. These findings reveal potential targets of FDp effectors and provide the first functional demonstration of variable pathogenicity between FDp strains, suggesting the need for future comparative genomic analyses of FDp strains as an important factor in exploring the management possibilities of FDp.

НЕКОТОРЫЕ АСПЕКТЫ СОЗДАНИЯ ОЗДОРОВЛЕНЫХ КОЛЛЕКЦИЙ ВИНОГРАДА АБОРИГЕННЫХ ДОНСКИХ СОРТОВ В КУЛЬТУРЕ IN VITRO

The article discusses some aspects of improving the elements of technology for creating of healthy collection of indigenous grape varieties in vitro. To recover from phytoplasma infec-tion, a chemotherapy method using the antibi-otic Cefotaxime has been developed. At high concentrations of the antibiotic, intoxication of plants and deterioration in their quality charac-teristics were noted. Plants of Makhrovatchik variety showed an improvement in survival rate at antibiotic concentrations of 100 and 200 mg/l: 64.3 and 60.7%, respectively. For long-term cultivation without replantings, the effectiveness of slowing down growth process-es by increasing the density of the nutrient me-dium was studied. The study was carried out on the varieties Makhrovatchik, Tsimlyansky Sergienko. Studied concentrations of agar-agar were (6, 8, 10, 12, 13 and 15 g/l). A decrease in the rate of growth processes was noted in all studied varieties, but the rate of the decrease was different.

Prevalence of a 'Candidatus Phytoplasma solani'-Related Strain Designated as New 16SrXII-P Subgroup over 'Candidatus Arsenophonus phytopathogenicus' in Sugar Beet in Eastern Germany.

Two phloem-limited pathogens, 'Candidatus Arsenophonus phytopathogenicus' and 'Candidatus Phytoplasma solani', threaten sugar beet production in France, Switzerland, and Germany. Previous studies of these pathogens in Germany had focused on its western and southern regions, leaving a knowledge gap about eastern Germany. Despite their importance, this study is the first to investigate phytoplasmas in sugar beet in Saxony-Anhalt, Germany. A phytoplasma strain related to 'Ca. P. solani' is found predominant in Saxony-Anhalt, unlike in France, where 'Ca. P. solani' has a minor role compared with 'Ca. A. phytopathogenicus'. The phytoplasma strain infecting sugar beet in Saxony-Anhalt was classified into a new subgroup designated as 16SrXII-P. The multilocus sequence analysis (MLSA) of nonribosomal genes of the novel phytoplasma strain showed that it is significantly different from the reference and all previously reported 'Ca. P. solani' strains including the strain from western Germany. Analyses of sugar beet samples from previous years confirmed the presence of the 16SrXII-P strain in sugar beet as early as 2020 and also in Bavaria in southern Germany. Based on 16S rDNA analysis, 'Ca. A. phytopathogenicus' in Saxony-Anhalt is identical to strains in sugar beet in other parts of Germany and France, as well as to a strain in potato from Germany. The presence and prevalence of two phytoplasmas in sugar beet in Germany suggest that more attention should be directed toward understanding phytoplasma infection in sugar beet in this country.

Novel Insights into Phytoplasma Effectors

Effectoromics has become integral to the identification of pathogen targets and/or host-resistant proteins for the genetic improvement of plants in agriculture and horticulture. Phytoplasmas are the causal agents of more than 100 plant diseases in economically important crops such as vegetables, spices, medicinal plants, ornamentals, palms, fruit trees, etc. To date, around 20 effectors in phytoplasmas have been experimentally validated but the list of putative effectors comprises hundreds of different proteins. Very few families (tribes) have been identified based on homology, such as the SAP05-like, SAP11-like, SAP54-like and TENGU-like families. The lack of conservation in amino acid sequences slows the progress of effectoromics in phytoplasmas since many effectors must be studied individually. Here, 717 phytoplasma effector candidates and 21 validated effectors were characterized in silico to identify common features. We identified functional domains in 153 effectors, while 585 had no known domains. The most frequently identified domain was the sequence-variable mosaic domain (SVM domain), widely distributed in 87 phytoplasma effectors. Searching for de novo amino acid motifs, 50 were found in the phytoplasma effector dataset; 696 amino acid sequences of effectors had at least 1 motif while 42 had no motif at all. These data allowed us to organize effectors into 15 tribes, uncovering, for the first time, evolutionary relationships largely masked by lack of sequence conservation among effectors. We also identified 42 eukaryotic linear motifs (ELMs) in phytoplasma effector sequences. Since the motifs are related to common functions, this novel organization of phytoplasma effectors may help further advance effectoromics research to combat phytoplasma infection in agriculture and horticulture.

Clarithromycin Suppresses Apple Proliferation Phytoplasma in Explant Cultures.

Apple proliferation, caused by 'Candidatus Phytoplasma mali', is one of the most important economic threats in the field of apple production. Especially at a young age, infected trees can be affected by excessive bud proliferation and general decline. The fruit quality is also significantly reduced by this disease. To investigate treatment options, we applied a clarithromycin chemotherapy to infected in vitro cultures of 'Golden Delicious'. With increasing concentrations of clarithromycin in the media, the phytoplasma load decreased rapidly after one month of treatment, but phytotoxicity led to a pronounced mortality at 40 mg/L, which was the highest dose used in our experiment. Out of 45 initial explants, we obtained one negative mericlone and two mericlones with a concentration of phytoplasma DNA at the detection limit of PCR. The culture propagated from the mericlone that tested negative remained phytoplasma-free after 18 months of subculturing. Our results suggest the applicability of macrolide antibiotics against phytoplasma infections in vitro; however, it might be challenging to find the threshold zone where the concentration is sufficient for pathogen elimination, but not lethal for the plant material of different cultivars.

Protective and curative effect of an experimental biocomplex against Flavescence dorée phytoplasma infection and transmission by Scaphoideus titanus under laboratory conditions

Flavescence dorée (FD) is one of the most devastating diseases threatening grapevine cultivation in Europe. The causal agents are phytoplasmas, which reside in phloem vessels of infected plants and in the body of leafhopper vectors, the most important being Scaphoideus titanus. Current control mainly relies on vector management, which is often insufficient to prevent from disease outbreaks. Suitable methods to interfere with phytoplasmas in planta are constantly sought to support insecticide sprays. We tested a biocomplex containing trace elements and soluble sugars, with potential systemic spread and antimicrobial activity, with the aim to verify its capability to penetrate plant phloem and to interfere with phytoplasma survival in vector-inoculated plants, using two FD isolates (FD-C and FD-D). The penetration into the plant phloem, as well as its acquisition by phloem-feeding vectors, were tested using an iridium-marked biocomplex. Phytoplasma infection was detected by real Time PCR and compared in plants exposed to biocomplex treatment before inoculation (short-time and mid-time preventive application), and to treatment after inoculation (curative application). We also assessed if the biocomplex interferes with the vector competence of S. titanus, by conducting phytoplasma transmission trials with sequential inoculation steps, where untreated and biocomplex-treated grapevine plantlets were sequentially offered to infectious leafhoppers. Foliar treatment with the biocomplex supported the penetration of phloem tissues, and the product was successfully acquired by the experimental phytoplasma vector Euscelidius variegatus after feeding on the phloem of treated plants. When the biocomplex was applied on grapevines, it induced a significant reduction of phytoplasma infection regardless of FD isolate, both as a preventive and curative treatment, indicating an antibacterial activity in the plant. Moreover, sequential inoculation experiments showed a decrease of S. titanus inoculation rates after vector exposure to a biocomplex-treated plant, suggesting a reduction of transmission efficiency that was higher using the FD-C rather than FD-D isolate. Our results indicate the tested biocomplex as a promising tool for the creation of anti-phytoplasma formulations to support vector management in FD containment.

Combined Transcriptomic and Metabolomic Analyses of Defense Mechanisms against Phytoplasma Infection in Camptotheca acuminata Decne

Camptotheca acuminata Witches’-broom disease (CaWB) is the most destructive disease affecting C. acuminata in China. Previous studies on CaWB have failed to clarify the incidence pattern in C. acuminata after infection with phytoplasma. The time interval between phytoplasma infection of C. acuminata and the onset of Witches’-broom symptoms in C. acuminata was very long. C. acuminata inoculated with CaWB showed leaf margin scorching symptoms at 4 weeks in inoculated leaves. At 16 weeks after infection (WAI), old leaves were shed, while new leaves showed a mild leaf margin scorch; at 28 WAI, typical symptoms appeared. Transcriptomic and metabolomic analyses of the three sampling periods revealed 194 differentially expressed genes, mainly enriched in MAPK signaling, plant–pathogen interaction, phenylpropanoid biosynthesis, starch and phenylpropanoid biosynthesis, and phenylpropanoid biosynthesis pathways. The expression of calcium-dependent protein kinase (CDPK), β Ketoacyl-CoA Synthase1/10 (KCS1/10), and WRKY22/29 genes in the plant–pathogen interaction pathway significantly increased, indicating that they may be key genes in the CaWB phytoplasma-mediated maintenance of ROS homeostasis. Moreover, isochlorogenic acid B, atractylenolide II, and 3-methoxybenzoic acid were found, which might serve as signaling or functional substances in the defense response. Our results provide novel insights into the pathogenesis of CaWB and the defense response of C. acuminata under the influence of phytoplasma. Additionally, we identified potential candidate genes related to the defense response of C. acuminata, laying the foundation for further research.

Walnut witches'-broom disease threatens butternut restoration efforts in Indiana.

Due to the devastating effects caused by butternut canker disease (BCD), efforts to protect the endangered butternut (Juglans cinerea) tree through resistance breeding has been a primary focus of forest restoration efforts. Walnut witches'-broom (WWB) disease poses a serious threat to these restoration efforts. This study sought to confirm the presence of the WWB disease phytoplasma, 'Candidatus phytoplasma pruni,' in butternut research plantings in Indiana using molecular methods and Sanger sequencing and identify butternut families affected by the disease. We also sought to better understand the incidence of the WWB's phytoplasma in asymptomatic trees and asymptomatic branches of symptomatic trees to better direct management decisions. The results of the Sanger sequencing confirmed the presence of the WWB phytoplasma in the butternut restoration plantings, the first confirmation in Indiana based on sequencing to our knowledge, in both symptomatic and some asymptomatic trees. In addition, the WWB phytoplasma was detected in asymptomatic branches of symptomatic trees, indicating that phytoplasma infection is not necessarily localized to symptomatic tissues in a tree. Trees with positive molecular confirmation of the WWB phytoplasma consisted of 23 different butternut families and one family of Japanese walnut (J. ailantifolia), which is considered to be one of the most susceptible species to WWB disease. Based on these findings, future studies should prioritize identifying the hybridity and pedigrees of families and their susceptibility to WWB disease to aid in butternut restoration efforts.

The Endoplasmic Reticulum Is a Key Battleground between Phytoplasma Aggression and Host Plant Defense.

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.

Candidate pathogenicity factor/effector proteins of 'Candidatus Phytoplasma solani' modulate plant carbohydrate metabolism, accelerate the ascorbate-glutathione cycle, and induce autophagosomes.

The pathogenicity of intracellular plant pathogenic bacteria is associated with the action of pathogenicity factors/effectors, but their physiological roles for most phytoplasma species, including 'Candidiatus Phytoplasma solani' are unknown. Six putative pathogenicity factors/effectors from six different strains of 'Ca. P. solani' were selected by bioinformatic analysis. The way in which they manipulate the host cellular machinery was elucidated by analyzing Nicotiana benthamiana leaves after Agrobacterium-mediated transient transformation with the pathogenicity factor/effector constructs using confocal microscopy, pull-down, and co-immunoprecipitation, and enzyme assays. Candidate pathogenicity factors/effectors were shown to modulate plant carbohydrate metabolism and the ascorbate-glutathione cycle and to induce autophagosomes. PoStoSP06, PoStoSP13, and PoStoSP28 were localized in the nucleus and cytosol. The most active effector in the processes studied was PoStoSP06. PoStoSP18 was associated with an increase in phosphoglucomutase activity, whereas PoStoSP28, previously annotated as an antigenic membrane protein StAMP, specifically interacted with phosphoglucomutase. PoStoSP04 induced only the ascorbate-glutathione cycle along with other pathogenicity factors/effectors. Candidate pathogenicity factors/effectors were involved in reprogramming host carbohydrate metabolism in favor of phytoplasma own growth and infection. They were specifically associated with three distinct metabolic pathways leading to fructose-6-phosphate as an input substrate for glycolysis. The possible significance of autophagosome induction by PoStoSP28 is discussed.

Physiological and Transcriptomic Analysis of ‘Candidatus Phytoplasma pruni’ Infection in Prunus persica

‘ Candidatus Phytoplasma pruni’ is the causative agent of X-disease on peach ( Prunus persica) trees. Infected trees exhibit premature yellowing, leaf necrosis causing a shot-hole appearance, limb dieback, and eventual death. How pathogen infection leads to these symptoms is unknown. This study undertook a modern characterization of the disease by assessing the physiological and transcriptomic consequences of phytoplasma infection. Phytoplasma titer was high in the symptomatic tissues and undetected or low in asymptomatic tissues. Symptomatic leaves had a significant decrease in chlorophyll a, chlorophyll b, and carotenoids. Transcriptomic analysis showed alterations in genes related to phytohormone synthesis and signaling, circadian rhythms, lignification, and sugar synthesis and transport. Several transcripts that might be related to symptom development were identified. Collectively, these data give a much clearer picture of symptom development in ‘ Ca. P. pruni’-infected P. persica and provide several avenues for further research in determining how ‘ Ca. P. pruni’ interacts with its host to elicit the observed symptoms. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Simultaneous detection and quantification by multiplex qPCR of 'Candidatus Phytoplasma asteris' and 'Candidatus Phytoplasma fraxini' in a plant host and insect vectors

AbstractPhytoplasmas are bacteria transmitted by insects that can cause plant diseases. In Bogotá 'Candidatus Phytoplasma asteris' and ' Candidatus Phytoplasma fraxini', infect 11 species of urban trees, weeds, grass, potato and strawberry. A set of primers, that amplify both phytoplasmas species were designed and used for absolute and relative qPCR quantification of the 16SrRNA gene. The primers AJ-16Sr-F/AJ-16Sr-R allowed the amplification of ‘Ca. P. asteris’, ‘Candidatus Phytoplasma palmae’, ‘Ca. P. fraxini’ and ‘Candidatus Phytoplasma phoenicium’, not of ‘Candidatus Phytoplasma pruni’. Absolute qPCR detected phytoplasmas between 1 × 109 and 1 × 103 copies/μL DNA extract. Two species-specific hydrolysis probes, AJ-16SrI-Cy5.5 and AJ-16SrVII-TexRed, were designed to detect 'Ca. P. asteris' and 'Ca. P. fraxini' respectively, using the AJ-16Sr-F/AJ-16Sr-R primers. For relative quantification, the 18SrRNA gene was used as normalizer. Relative qPCR detected phytoplasmas between 1 × 109 and 1 × 103 copies/μL DNA extract. Multiplex reactions allowed the specific quantification of 'Ca. P. asteris', 'Ca. P. fraxini' in comparison to the normalizer. qPCR methods were validated on natural hosts Andean oak trees and leafhoppers. The relative quantification values were higher for 'Ca. P. fraxini' (x̅ RQ = 3203.1 ± 2622,9 n = 14) compared with 'Ca. P. asteris' (x̅ RQ = 14.9 ± 24,5 n = 6) in oak tree samples. In the leafhoppers, the relative quantification values ranged between RQ = 26.5 and RQ = 294,927.3 for 'Ca. P. fraxini’ and RQ = 34.8 and RQ = 1722.2 for 'Ca. P. asteris'. In conclusion, although absolute qPCR allowed the quantification of phytoplasmas by comparing Cq (quantification cycle) values of samples with a standard curve, it did not allow to differentiate between 'Ca. P. asteris' and 'Ca. P. fraxini'. In contrast, relative qPCR assays using specific hydrolysis probes allowed the specific detection and quantification of each phytoplasma, in individual and mixed infections in insect vectors and plant hosts.

Sweet Potato (Ipomea batatas L.): A New Alternative Host of Two Phytoplasmas Associated with Coconut Lethal Yellowing Disease in Côte d’Ivoire

Aims: Coconut Lethal Yellowing Disease has been threatening coconut plantations in Côte d'Ivoire for ten years and has destroyed more than 400 ha. The destruction of the coconut palm leads to phytoplasmas conservation on other plants grown in association or near of the disease outbreaks. Thus, the search for possible alternative host species was conducted.
 Methodology: Surveys were carried out in infected coconut plantations, in order to describe symptoms associated with phytoplasma infections and to collect leaf samples of plant species other than coconut. Symptomatic and non-symptomatic leaves of sweet potato (Ipomea batatas L.) were collected for DNA extraction. The extracted DNA was subjected to molecular characterization.
 Results: Mosaic and leaf reduction symptoms were observed on sweet potato leaves. From these samples, two phytoplasma strains were associated. Molecular analysis showed the presence of the endemic phytoplasma strain belonging to the 16SrXXII-B subgroup ʺCandidatus Phytoplasma palmicolaʺ. Sequencing and phylogenetic analysis also revealed the presence of a new phytoplasma strain, sharing 99% similarity with 16SrIV group strains ʺCandidatus Phytoplasma palmaeʺ.
 Conclusion: The detection of these strains confirms sweet potato for the very first time, as an alternative host for coconut lethal yellowing phytoplasmas in Côte d'Ivoire.

First report of 'Candidatus Phytoplasma rubi' associated with Rubus stunt disease of raspberry and blackberry in the Czech Republic.

Raspberries (Rubus idaeus L.), occurring in the temperate zone of the northern hemisphere and blackberries (R. fruticosus L.), cultivated and growing all over the world, are plant species of the family Rosaceae. These species are susceptible to phytoplasma infections, which cause Rubus stunt disease. It spreads uncontrolled by vegetative propagation of plants (Linck and Reineke 2019a) and by phloem-sucking insect vectors, especially Macropsis fuscula (Hemiptera: Cicadellidae) (de Fluiter and van der Meer, 1953; Linck and Reineke 2019b). During a survey in commercial field in June 2021, over 200 raspberry bushes cv Enrosadira exhibiting typical symptoms of Rubus stunt were observed in Central Bohemia. Symptoms included dieback, leaf yellowing/reddening, stunted growth, severe phyllody and fruit malformations. Most diseased plants were growing in the edge rows of the field (about 80%). No symptomatic plants were observed in the middle of the field. Similar symptoms were observed in private gardens in South Bohemia on raspberry cv Rutrago and blackberry (unknown cultivar) in June 2018 and August 2022, respectively. DNA was extracted using the DNeasy Plant Mini Kit (Qiagen GmbH, Hilden, Germany) from flower stems and parts affected by phyllody of seven symptomatic plants as well as flower stems, leaf midribs, and petioles of five asymptomatic field plants. The DNA extracts were analyzed by a nested polymerase chain reaction assay using universal phytoplasma P1A/P7A primers followed by R16F2m/R1m and the group-specific R16(V)F1/R1 primers (Bertaccini et al. 2019). All samples from the symptomatic plants yielded an amplicon of expected size, while no product was amplified in asymptomatic plants. The P1A/P7A amplicons from three selected plants (two raspberries and one blackberry, each from different location) were cloned and bi-directionally Sanger sequenced (GenBank Accession Nos.OQ520100-2). The sequences spanned nearly full-length of 16S rRNA gene, 16S-23S rRNA intergenic spacer, tRNA-Ile gene, and a partial 23S rRNA gene. BLASTn search revealed the highest sequence identity (99.8-99.9%, query coverage 100%) to 'Candidatus Phytoplasma rubi' strain RS (GenBank Accession No. CP114006). To further characterize the 'Ca. P. rubi' strains, all these three samples were subjected to multigene sequence analysis. Sequences from a major portion of the tuf, rplV-rpsC, rpsH-rplR, uvrB-degV, and rplO-SecY-map genes (Acc. Nos. OQ506112-26) were obtained as described previously (Fránová et al. 2016). Comparison to GenBank sequences confirmed their highest identity (99.6-100%, query coverage 100%) with 'Ca. P. rubi' RS strain, regardless of their geographic location and host (raspberry or blackberry). Recently, Bertaccini et al. (2022) suggested the 98,65 % 'Ca. Phytoplasma' strain identity threshold within 16Sr RNA sequences. In this survey, all three strains sequenced shared ≥99.73% sequence identity of the analysed 16S rRNA gene sequences and the high identity in the other genes with the reference 'Ca. P. rubi' RS strain. To our knowledge, this is the first report of Rubus stunt disease in the Czech Republic as well as the first molecular identification and characterization of 'Ca. P. rubi' from raspberry and blackberry in our country. As Rubus stunt disease is of great economic importance (Linck and Reineke 2019a), the pathogen detection and prompt removal of the diseased shrubs are essential to mitigating the spread and impact of the disease.

First Report of Candidatus Phytoplasma asteris (16SrI-B subgroup) associated with phyllody disease of fenugreek in the world.

Fenugreek (Trigonella foenum-graecum) is a leafy vegetable and spice crop, native to Indian subcontinent and Eastern Mediterranean region. Phytoplasma infection symptoms were observed in fenugreek at ICAR-National Bureau of Plant Genetic Resources Regional Station, Jodhpur and Agricultural Research Station Mandore Jodhpur, Rajasthan, India. The first appearance of phytoplasma suspected symptoms of little leaf was recorded after 50 days of sowing in the months of January 2022. The major symptoms recorded were virescence, phyllody, shoot proliferation, witches-broom, little leaf, yellowing and overall stunted growth in 146 germplasm accessions at NBPGR research farm, Jodhpur and one major commercially cultivated variety RMT 305 at Mandore Jodhpur. Ten samples from symptomatic and five samples from asymptomatic fenugreek plants were collected and processed for total DNA extraction using the Qiagen DNeasy plant mini kit (Germany). The extracted DNA was amplified using nested PCR assays with universal phytoplasma detection primers for 16S rRNA gene (P1/P7 and R16F2n/R16R2) and secA gene specific primers (SecAfor1/SecArev3 and SecAfor2/SecArev3) (Schneider et al. 1995; Gundersen and Lee 1996; Hodgetts et al. 2008). The amplicons of ∼1.25 kb with 16S rRNA and ∼480 bp with secA gene specific primers were amplified in all symptomatic fenugreek samples. In negative control (asymptomatic plants) no amplification was observed with either of gene specific primers in gel electrophoresis. PCR amplified products from the six selected positive samples (FPP-NBPGR-J-01 to FPP-NBPGR-J-04 and FPP-MND-01 to FPP-MND-02) of 16S rRNA and secA gene, were sequenced from both ends. Sequences were deposited in the NCBI GenBank with accession numbers ON756108-ON756113 for 16S rRNA gene sequences and ON745809 to ON745814 for secA gene sequences. BLAST analysis of 16S rRNA and secA sequences revealed 100% sequence identity among themselves and 99.95 to 100% sequence identity with the earlier reported phytoplasma strains of aster yellows group related phytoplasma strains (GenBank Acc. No. MN239504, MN080270) belonging to Ca. P. asteris (16SrI group). Further analyses of the 16S rRNA and secA gene-based phylogenetic tree and the iPhyClassifier-based virtual RFLP analysis of 16S rRNA gene study demonstrated that the phytoplasma associated with fenugreek phyllody belonged to 16Sr group I ('Ca. P. asteris') and subgroup B (GenBank accession AP006628), with similarity coefficient of 1.0. Earlier association of 16Sr-II-D subgroup (Ca. P. australasiae) with fenugreek as host was reported from Pakistan (Malik et al., 2020). To the best of our knowledge, this is the first report of a 'Ca. P. asteris', 16SrI-B subgroup related phytoplasma strain associated with fenugreek phyllody in the world. The 16SrI-B phytoplasma strain is a widely distributed strain associated with several agricultural and horticultural crops of India (Rao 2021). This is not only the first instance of fenugreek phyllody disease found in India, but also the first instance of fenugreek phyllody caused by 16SrI-B subgroup phytoplasma worldwide. This report has epidemiological significance and needs immediate attention, as fenugreek is one of the most common seed spice crop being grown all over India.

Genome-Wide Characterization of Calmodulin and Calmodulin-like Protein Gene Families in Paulownia fortunei and Identification of Their Potential Involvement in Paulownia Witches' Broom.

As significant Ca2+ sensors, calmodulin (CaM) and calmodulin-like proteins (CML), have been associated with a variety of environmental conditions in plants. However, whether CaMs/CMLs are related to the stress of phytoplasma infection has not been reported in Paulownia fortunei. In the current study, 5 PfCaMs and 58 PfCMLs were detected through a genome-wide investigation. The number of EF-hand motifs in all PfCaMs/CMLs varied. Bioinformatics analyses, including protein characteristics, conserved domain, gene structure, cis-elements, evolutionary relationship, collinearity, chromosomal location, post-translation modification site, subcellular localization and expression pattern analyses, represented the conservation and divergence of PfCaMs/CMLs. Furthermore, some PfCaMs/CMLs might be involved in plants' reaction to phytoplasma infection and exogenous calcium therapy, indicating these genes may play a role in abiotic as well as biotic stress responses. In addition, subcellular localization analysis showed that PfCML10 was located in the cell membrane and nucleus. In summary, these findings establish a stronger platform for their subsequent functional investigation in trees and further characterize their roles in Paulownia witches' broom (PaWB) occurrence.

Jujube witches' broom phytoplasmas inhibit ZjBRC1-mediated abscisic acid metabolism to induce shoot proliferation.

Jujube witches' broom (JWB) phytoplasmas parasitize the sieve tubes of diseased phloem and cause an excessive proliferation of axillary shoots from dormant lateral buds to favour their transmission. In previous research, two JWB effectors, SJP1 and SJP2, were identified to induce lateral bud outgrowth by disrupting ZjBRC1-mediated auxin flux. However, the pathogenesis of JWB disease remains largely unknown. Here, tissue-specific transcriptional reprogramming was examined to gain insight into the genetic mechanisms acting inside jujube lateral buds under JWB phytoplasma infection. JWB phytoplasmas modulated a series of plant signalling networks involved in lateral bud development and defence, including auxin, abscisic acid (ABA), ethylene, jasmonic acid, and salicylic acid. JWB-induced bud outgrowth was accompanied by downregulation of ABA synthesis within lateral buds. ABA application rescued the bushy appearances of transgenic Arabidopsis overexpressing SJP1 and SJP2 in Col-0 and ZjBRC1 in the brc1-2 mutant. Furthermore, the expression of ZjBRC1 and ABA-related genes ZjHB40 and ZjNCED3 was negatively correlated with lateral main bud outgrowth in decapitated healthy jujube. Molecular evidence showed that ZjBRC1 interacted with ZjBRC2 via its N-terminus to activate ZjHB40 and ZjNCED3 expression and ABA accumulation in transgenic jujube calli. In addition, ZjBRC1 widely regulated differentially expressed genes related to ABA homeostasis and ABA signalling, especially by binding to and suppressing ABA receptors. Therefore, these results suggest that JWB phytoplasmas hijack the ZjBRC1-mediated ABA pathways to stimulate lateral bud outgrowth and expansion, providing a strategy to engineer plants resistant to JWB phytoplasma disease and regulate woody plant architecture to promote crop yield and quality.

Epidemiology of flavescence dorée and hazelnut decline in Slovenia: geographical distribution and genetic diversity of the associated 16SrV phytoplasmas.

Flavescence dorée (FD) phytoplasma from 16SrV-C and -D subgroups cause severe damage to grapevines throughout Europe. This phytoplasma is transmitted from grapevine to grapevine by the sap-sucking leafhopper Scaphoideus titanus. European black alder and clematis serve as perennial plant reservoirs for 16SrV-C phytoplasma strains, and their host range has recently been extended to hazelnuts. In Slovenia, hazelnut orchards are declining due to 16SrV phytoplasma infections, where large populations of the non-autochthonous leafhopper Orientus ishidae have been observed. To better characterise the phytoplasma-induced decline of hazelnut and possible transmission fluxes between these orchards and grapevine, genetic diversity of 16SrV phytoplasmas in grapevine, hazelnut and leafhoppers was monitored from 2017 to 2022. The nucleotide sequence analysis was based on the map gene. The most prevalent map genotype in grapevine in all wine-growing regions of Slovenia was M54, which accounted for 84% of the 176 grapevines tested. Besides M54, other epidemic genotypes with lower frequency were M38 (6%), M51 (3%), M50 (2%) and M122 (1%). M38, M50 and M122 were also detected in infected cultivated hazelnuts and in specimens of O. ishidae leafhopper caught in declining hazelnut orchards. It suggests that this polyphagous vector could be responsible for phytoplasma infection in hazelnut orchards and possibly for some phytoplasma exchanges between hazelnuts and grapevine. We hereby describe new genotypes: M158 in grapevine as well as four never reported genotypes M159 to M162 in hazelnut. Of these four genotypes in hazelnut, one (M160) was also detected in O. ishidae. Analysis of additional genes of the new genotypes allowed us to assign them to the VmpA-III cluster, which corresponds to the 16SrV-C strains previously shown to be compatible with S. titanus transmission.

N6‐methyladenosine modification changes during the recovery processes for Paulownia witches' broom disease under the methyl methanesulfonate treatment

AbstractPhytoplasmas induce diseases in more than 1000 plant species and cause substantial ecological damage and economic losses, but the specific pathogenesis of phytoplasma has not yet been clarified. N6‐methyladenosine (m6A) is the most common internal modification of the eukaryotic Messenger RNA (mRNA). As one of the species susceptible to phytoplasma infection, the pathogenesis and mechanism of Paulownia has been extensively studied by scholars, but the m6A transcriptome map of Paulownia fortunei (P. fortunei) has not been reported. Therefore, this study aimed to explore the effect of phytoplasma infection on m6A modification of P. fortunei and obtained the whole transcriptome m6A map in P. fortunei by m6A‐seq. The m6A‐seq results of Paulownia witches' broom (PaWB) disease and healthy samples indicate that PaWB infection increased the degree of m6A modification of P. fortunei. The correlation analysis between the RNA‐seq and m6A‐seq data detected that a total of 315 differentially methylated genes were predicted to be significantly differentially expressed at the transcriptome level. Moreover, the functions of PaWB‐related genes were predicted by functional enrichment analysis, and two genes related to maintenance of the basic mechanism of stem cells in shoot apical meristem were discovered. One of the genes encodes the receptor protein kinase CLV2 (Paulownia_LG2G000076), and the other gene encodes the homeobox transcription factor STM (Paulownia_LG15G000976). In addition, genes F‐box (Paulownia_LG17G000760) and MSH5 (Paulownia_LG8G001160) had exon skipping and mutually exclusive exon types of alternative splicing in PaWB‐infected seedling treated with methyl methanesulfonate, and m6A modification was found in m6A‐seq results. Moreover, Reverse Transcription–Polymerase Chain Reaction (RT‐PCR) verified that the alternative splicing of these two genes was associated with m6A modification. This comprehensive map provides a solid foundation for revealing the potential function of the mRNA m6A modification in the process of PaWB. In future studies, we plan to verify genes directly related to PaWB and methylation‐related enzymes in Paulownia to elucidate the pathogenic mechanism of PaWB caused by phytoplasma invasion.