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Abstract Background Little leaf disease caused by phytoplasma infection is a significant threat to eggplant (also known as brinjal) cultivation in India. This study focused on the molecular characterisation of the phytoplasma strains and insect vectors responsible for its transmission and screening of brinjal germplasm for resistance to little leaf disease. Results Surveys conducted across districts in the Tamil Nadu state of India during 2021–2022 showed a higher incidence of phytoplasma during the Zaid (March to June), followed by Kharif (June to November) and Rabi (November to March) seasons with mean incidence ranging from 22 to 27%. As the name indicates, phytoplasma infection results in little leaf (reduction in leaf size), excessive growth of axillary shoots, virescence, phyllody, stunted growth, leaf chlorosis and witches’ broom symptoms. PCR amplification with phytoplasma-specific primers confirmed the presence of this pathogen in all symptomatic brinjal plants and in Hishimonus phycitis (leafhopper), providing valuable insights into the role of leafhoppers in disease transmission. BLAST search and phylogenetic analysis revealed the phytoplasma strain as “Candidatus Phytoplasma trifolii”. Insect population and disease dynamics are highly influenced by environmental factors such as temperature, relative humidity and rainfall. Further, the evaluation of 22 eggplant accessions revealed immune to highly susceptible responses where over 50% of the entries were highly susceptible. Finally, additive main effect and multiplicative interaction (AMMI) and won-where biplot analyses identified G18 as a best-performing accession for little leaf resistance due to its consistent responses across multiple environments. Conclusions This research contributes essential information on little leaf incidence, symptoms, transmission and resistance profiles of different brinjal genotypes, which together ensure effective and sustainable management of this important disease of eggplants.

As hemp (Cannabis sativa) emerges as a commercial crop in Nevada, mild to severe abnormal growth has been observed from many plants in commercial fields throughout the growing season. Affected plants exhibited a group of symptoms typically including stunting, leaf yellowing, excessive apical branching, clusters of witches’ broom, leaf rolling upwards, and leaf mottling and mosaic. These symptoms mostly showed up together in a plant or a crop and are defined here as abnormal growth syndrome (AGS). Between 2017 and 2019, the Nevada State Plant Pathology and Molecular Diagnostic Lab received 67 symptomatic hemp samples from Nevada for diagnosis, and ‘Candidatus Phytoplasma trifolii’ was detected in 14 samples (21%). To investigate additional biotic agents associated with AGS, PCR products generated by primers P1/P7 were cloned into a pGEM-T vector and sequenced, and Spiroplasma citri DNA was found in two samples (3%). All 67 DNA samples were further tested for beet curly top virus (BCTV), and 57 samples (85%) were found to be infected by BCTV. Twelve samples (18%) were coinfected by both ‘Ca. Phytoplasma trifolii’ and BCTV, and two (3%) were coinfected by S. citri, ‘Ca. Phytoplasma trifolii’, and BCTV. The findings suggest that BCTV is the most prevalent pathogen causing the hemp abnormal growth in Nevada, but ‘Ca. Phytoplasma trifolii’ and S. citri may also contribute to the severity and complexity of symptoms. Thus, hemp abnormal growth can be attributed to single, dual, or triple infections of these three leafhopper-vectored mollicutes and virus. [Figure: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Cacopsylla mali, C. melanoneura, C. picta, C. pruni, C. pyri, and C. pyrisuga belong among important pests of fruit trees. They harm trees by sucking and transmitting pathogenic bacteria of the ‘Candidatus Phytoplasma’, 16SrX group, which leads to declining and death of fruit trees. Protection against these pests depends on their early reliable identification and monitoring in fruit nurseries and orchards. In general, the determination of adult psyllids is based on morphological criteria, however, it is difficult or even impossible to perform determination using eggs and nymphs. In this study, a recently developed real-time PCR method validated for the determination of adult psyllids, was applied for the identification of other developmental stages. A total, 46 samples of eggs and nymph instars N1 to N5 were successfully determined, indicating the PCR identification system can be reliably used also for these types of specimens. Specific PCRs detected three species – C. melanoneura, C. pyri a C. pruni which were also confirmed by sequencing of the cytochrome c oxidase subunit I (COI) gene. Thus, this innovative molecular method can be used for a quick and routine determination of selected Cacopsylla species infecting fruit trees in all developmental stages.

Coconut (Cocos nucifera L.) is an important palm species that serves as the mainstay of several industries and contributes to the livelihoods of millions of smallholder farmers. International exchange of coconut germplasm has been undertaken for several decades to facilitate the conservation of selected varieties within global genebanks and for the distribution to farmers and scientists. In vitro systems are a convenient and an efficient method for the exchange of coconut germplasm. However, it is possible that these tissue culture systems can transfer lethal pathogens causing a threat to the importing countries. In this review, the following topics are discussed: the major disease-causing agents of concern, the various tissues that could be used for coconut germplasm exchange, and the techniques available for the detection and elimination of disease-causing agents from various transmission systems. Additionally, the lack of clear, science-backed guidelines to facilitate the exchange of in vitro coconut materials is raised, along with recommendations for future studies to ensure the safe movement of coconut germplasm without biosecurity risks.

Sugarcane white leaf (SCWL) disease, caused by Candidatus Phytoplasma sacchari, results in the most damage to sugarcane plantations. Some SCWL canes can grow unnoticed through the maturation phase, subsequently resulting in an overall low sugar yield, or they can be used accidentally as seed canes. In this work, 12-month-old SCWL and asymptomatic canes growing in the same field were investigated. An abundance of phytoplasma in SCWL canes affected growth and sugar content as well as alterations of transcriptomic profiles corresponding to several pathways that responded to the infection. Suppression of photosynthesis, porphyrin and chlorophyll metabolism, coupled with an increase in the expression of chlorophyllase, contributed to the reduction in chlorophyll levels and photosynthesis. Blockage of sucrose transport plausibly occurred due to the expression of sugar transporters in leaves but suppression in stalks, resulting in low sugar content in canes. Increased expression of genes associated with MAPK cascades, plant hormone signaling transduction, callose plug formation, the phenylpropanoid pathway, and calcium cascades positively promoted defense mechanisms against phytoplasma colonization by an accumulation of lignin and calcium in response to plant immunity. Significant downregulation of CPK plausibly results in a reduction in antioxidant enzymes and likely facilitates pathogen invasion, while expression of sesquiterpene biosynthesis possibly attracts the insect vectors for transmission, thereby enabling the spread of phytoplasma. Moreover, downregulation of flavonoid biosynthesis potentially intensifies the symptoms of SCWL upon challenge by phytoplasma. These SCWL sugarcane transcriptomic profiles describe the first comprehensive sugarcane–phytoplasma interaction during the harvesting stage. Understanding molecular mechanisms will allow for sustainable management and the prevention of SCWL disease—a crucial benefit to the sugar industry.

Abstract Phytoplasmas are obligate cell wall-less prokaryotic bacteria that primarily multiply in plant phloem tissue. Jujube witches’ broom (JWB) associated with phytoplasma is a destructive disease of jujube (Ziziphus jujuba Mill.). Here we report the complete ‘Candidatus Phytoplasma ziziphi’ chromosome of strain Hebei-2018, which is a circular genome of 764,108-base pairs with 735 predicted CDS. Notably, extra 19,825 bp (from 621,995 to 641,819 bp) compared to the previously reported one complements the genes involved in glycolysis, such as pdhA, pdhB, pdhC, pdhD, ackA, pduL and LDH. The synonymous codon usage bias (CUB) patterns by using comparative genomics analysis among the 9 phytoplasmas were similar for most codons. The ENc-GC3s analysis among the 9 phytoplasmas showed a greater effect under the selection on the CUBs of phytoplasmas genes than mutation and other factors. The genome exhibited a strongly reduced ability in metabolic synthesis, while the genes encoding transporter systems were well developed. The genes involved in sec-dependent protein translocation system were also identified.The expressions of nine FtsHs encoding membrane associated ATP-dependent Zn proteases and Mn-SodA with redox capacity in the Ca. P. ziziphi was positively correlated with the phytoplasma concentration. Taken together, the genome will not only expand the number of phytoplasma species and provide some new information about Ca. P. ziziphi, but also contribute to exploring its pathogenic mechanism.

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.

Abstract Phytoplasmas 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.

Flavescence dorée (FD) has been recorded in French vineyards since the mid-1950s; it has rapidly become a major threat to vineyard survival in different European grape-growing areas. Infection by the phytoplasma ‘Candidatus Phytoplasma vitis’ causes leaf chlorosis and disturbs primary and secondary core metabolic pathways; it can also occlude sieve plates, therefore affecting normal solute transport through the phloem. Thus, in the present study we hypothesised that the metabolomic fingerprint of the phloem sap changes in FDp-infected vines. The metabolome of the phloem sap collected by centrifugation from the bark of the stem of healthy and infected vines (cv. ‘Loureiro’) was profiled by ESI-HRMS at two snapshot time points: E-L 33 (grape berry still hard and green) and E-L 38 (grape berry at maturity). An untargeted analysis of the phloem sap enabled the identification of 476 metabolites, whereas the targeted analysis focused on eight main classes, namely acids, sugar and polyols, amino acids, phenolic acids, hormones, vitamins and N compounds, as well as on several minor classes like fatty acids, phenols or diols. Depending on the developmental stage of the vine, major differences were observed in the composition of cv. ‘Loureiro’ phloem sap in response to FD infection in terms of sugar, organic acid and amino acid content; furthermore, compounds involved in plant defense, such as salicylic acid, ABA, vitamin C and gallic acid also significantly changed. Overall, these results have contributed towards increasing knowledge about the complex interactions between vine and FD phytoplasma.

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.

Cassava witches’ broom disease (CWBD) is one of the main diseases of cassava in Southeast Asia (SEA). Affected cassava plants show reduced internodal length and proliferation of leaves (phyllody) in the middle and top part of the plant, which results in reduced root yields of 50% or more. It is thought to be caused by phytoplasma; however, despite its widespread distribution in SEA still little is known about CWBD pathology. The overarching goal of this study was to review and corroborate published information on CWBD biology and epidemiology considering recent field observations. We report the following: (1) CWBD symptoms are conserved and persistent in SEA and are distinct from what has been reported as witches’ broom in Argentina and Brazil. (2) In comparison with cassava mosaic disease, another major disease of cassava in SEA, symptoms of CWBD develop later. (3) Phytoplasma detected in CWBD-affected plants belong to different ribosomal groups and there is no association study available indicating phytoplasma as the causing agent of CWBD. These findings are essential clues for designing surveillance and management strategies and for future studies to better understand the biology, tissue localization and spatial spread of CWBD in SEA and other potential risk areas.

Haplaxius crudus is an economically important species of cixiid planthopper that is widespread and abundant throughout the Caribbean basin. It is the vector of lethal yellowing and putative vector of lethal bronzing, both phytoplasma diseases of palm that cause death in Florida and the Caribbean. The primary objective of this study was to evaluate the genetic diversity of H. crudus in Florida to determine whether divergent populations existed. The 5′ region of the cytochrome c oxidase subunit I gene was used as the molecular marker. DNA sequences were obtained from 236 specimens collected throughout Florida, United States as well as populations from the southeastern United States. Populations from Costa Rica, Colombia, and Jamaica were included to compare differences between isolated populations. In Florida, four haplotypes were discovered, with 97% of individuals belonging to a single haplotype, two smaller haplotypes comprising six and four individuals, and a single haplotype comprising one individual. Populations from Texas and Mississippi represented distinct haplotypes whereas populations from Georgia and South Carolina were identical to the predominant haplotype in Florida. Populations from Costa Rica and Colombia were highly divergent whereas the population from Jamaica was 100% identical to the predominant population in Florida. These findings highlight measurable levels of genetic variability of H. crudus in Florida, and the similarity to populations from Jamaica highlight the need for more robust sampling throughout the Caribbean to better understand movement and invasion potential of this species.[Figure: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Two unrelated plant species, green pea and parthenium weed, harboring typical phytoplasma symptoms, were discovered in Yunlin, Taiwan. Green pea (Pisum sativum.) and parthenium weed (Parthenium hysterophorus L.) are both herbaceous annual plants belonging to the Fabaceae and Asteraceae families, respectively. Displayed symptoms were witches’ broom, phyllody and virescence, which are typical indications of phytoplasma infection. Pleomorphic phytoplasma-like bodies were observed under the transmission electron microscope in the sieve elements of symptomatic green pea and parthenium weed. The iPhyClassifier-based virtual RFLP study demonstrated that the phytoplasma associated with the diseased plants belongs to the 16SrII-V subgroup. The disease symptoms of both plants can be explained by the identification of PHYL1 and SAP11 effectors, identical to those of peanut witches’ broom phytoplasma. The phytoplasma strains identified in this study present a very close phylogenetic relationship with other 16SrII-V subgroup phytoplasma strains discovered in Taiwan. These results not only convey the local status of the 16SrII-V subgroup phytoplasma strains but also encourage attention to be given to preventing the spread of this threat before it becomes pervasive.

Sisal purple leafroll disease (SPLD) is currently the most destructive disease affecting sisal in China, yet its aetiology remains unclear. In our previous research, it was verified to be associated with phytoplasmas, and nested PCR based on the 16S rRNA gene using universal primers R16mF2/R16mR1 followed by R16F2n/R16R2 was confirmed as the most effective molecular method for the detection of phytoplasmas associated with SPLD (SPLDaP). However, the method has a shortcoming of inaccuracy, for it could produce false positive results. To further manage the disease, accurate detection is needed. In this study, we developed a specific nested PCR assay using universal primers R16F2n/R16R2, followed by a set of primers designed on 16Sr gene sequences amplified from SPLDaP, nontarget bacteria from sisal plants, and other phytoplasma subgroups or groups. This established method is accurate, specific, and effective for detection of 16SrI group phytoplasma in sisal, and its sensitivity is up to 10 fg/μL of total DNA. It also minimized the false positive problem of nested PCR using universal primers R16mF2/R16mR1 followed by R16F2n/R16R2. This method was further used to verify the presence of phytoplasma in Dysmicoccusneobrevipes, and the results showed that D. neobrevipes could be infected by SPLDaP and thus could be a candidate for vector transmission assays.

While host gene expression has been related to symptoms associated with different phytoplasma diseases, it is unknown why some phytoplasmas are associated with different symptoms in genotypes of the same plant species. Pear tree selections showed symptoms of either leaf reddening (selection 8824-1) or leaf curling (selection 9328-1) associated with pear decline (PD) phytoplasma presence. PD populations were similar in leaves and shoots of the two selections, but in the roots, populations were significantly lower in selection 8824-1 than in 9328-1, indicating greater resistance. For host carbohydrate metabolism gene expression in PD-infected tissues, significant up-regulation in selection 8824-1 was observed for a sucrose synthase gene in leaves and an acid invertase gene in leaves and roots. These features have been associated with localized higher sugar levels in phytoplasma-infected tissues, and thus may be related to leaf reddening. For host stress/defense response gene expression in PD-infected tissues, significant up-regulation of a phenylalanine ammonia lyase gene was observed in PD-infected shoots of both selections; however, up-regulation of alcohol dehydrogenase gene in shoots, a chitinase gene in all tissues and a phloem protein 2 gene in roots was only observed for selection 8824-1. These changes indicate greater triggered innate immunity in roots associated with lower PD populations and leaf reddening. Leaf reddening may be related to changes in gene expression associated with increased sugar levels in leaves and stronger immune responses in several tissues, while leaf curling may be due to water stress resulting from dysfunctional root associated with higher PD populations in the roots.

Phytoplasmas are obligate cell-wall-less plant pathogenic bacteria that infect many economically important crops, causing considerable yield losses worldwide. Very little information is known about phytoplasma–host plant interaction mechanisms and their influence on sesame yield and oil quality. Therefore, our aim was to explore the ultrastructural and agro-physio-biochemical responses of sesame plants and their effects on sesame productivity and oil quality in response to phytoplasma infection. Sesame leaf samples exhibiting phyllody symptoms were collected from three experimental fields during the 2021 growing season. Phytoplasma was successfully detected by nested- polymerase chain reaction (PCR) assays using the universal primer pairs P1/P7 and R16F2n/R16R2, and the product of approximately 1200 bp was amplified. The amplified product of 16S rRNA was sequenced and compared with other available phytoplasma’s 16S rRNA in the GenBank database. Phylogenetic analysis revealed that our Egyptian isolate under accession number MW945416 is closely related to the 16SrII group and showed close (99.7%) identity with MH011394 and L33765.1, which were isolated from Egypt and the USA, respectively. The microscopic examination of phytoplasma-infected plants revealed an observable deterioration in tissue and cell ultrastructure. The primary and secondary metabolites considerably increased in infected plants compared with healthy ones. Moreover, phytoplasma-infected plants showed drastically reduced water content, chlorophyll content, growth, and yield components, resulting in 37.9% and 42.5% reductions in seed and oil yield, respectively. The peroxide value of the infected plant’s oil was 43.2% higher than that of healthy ones, suggesting a short shelf-life. Our findings will provide a better understanding of the phyllody disease pathosystem, helping us to develop effective strategies for overcoming such diseases.

The Northern Australia Quarantine Strategy (NAQS) is a biosecurity initiative operated by the Australian federal government’s Department of Agriculture, Water and the Environment (DAWE). It is unique worldwide because it deals specifically with the potential arrival via unregulated pathways of exotic threats from overseas in a vast and sparsely populated region. It aims to protect the nation’s animal- and plant-based production industries, as well as the environment, from incursions of organisms from countries that lie immediately to the north. These are diseases, pests, and weeds present in these countries that are currently either absent from, or under active containment in, Australia and may arrive by natural or human-assisted means. This review article focuses on the plant viruses and virus-like diseases that are most highly targeted by the NAQS program. It presents eight pathogen species/group entries in the NAQS A list of target pathogens, providing an overview of the historical and current situation, and collates some new data obtained from surveillance activities conducted in northern Australia and collaborative work overseas.

Bois Noir is a major yellows disease in grapevine with no current means of control. The endophyte Frateuria defendens (Frd), isolated from the insect vector of Bois Noir, is a potential biocontrol agent for this disease. The aim of this study was to determine an efficient way to introduce Frd into mature vines under field conditions. Shoots of young and field-grown vines were sprayed with Frd suspension supplemented with either Tween 20, BB5, DX, or Triton X-100 as surfactants. The presence of Frd was confirmed by PCR in sprayed leaves, and in leaves below and above the sprayed site. The results showed that cells of Frd penetrate and move inside the vine shoots under field conditions. Highest penetration rate was achieved when leaves were sprayed with 108 or 109 colony forming units per mL (CFU mL-1) of Frd with addition of 0.1% Tween 20. The addition of a surfactant is essential to increase the proportion of shoots with Frd.

Understanding temporal biological phenomena is a challenging task that can be approached using network analysis. Here, we explored whether network reconstruction can be used to better understand the temporal dynamics of bois noir, which is associated with ‘Candidatus Phytoplasma solani’, and is one of the most widespread phytoplasma diseases of grapevine in Europe. We proposed a methodology that explores the temporal network dynamics at the community level, i.e., densely connected subnetworks. The methodology offers both insights into the functional dynamics via enrichment analysis at the community level, and analyses of the community dissipation, as a measure that accounts for community degradation. We validated this methodology with cases on experimental temporal expression data of uninfected grapevines and grapevines infected with ‘Ca. P. solani’. These data confirm some known gene communities involved in this infection. They also reveal several new gene communities and their potential regulatory networks that have not been linked to ‘Ca. P. solani’ to date. To confirm the capabilities of the proposed method, selected predictions were empirically evaluated.

The present study examined the effects of Candidatus Phytoplasma solani infection on antioxidative metabolism in leaves and roots of carrot (Daucus carota L.). Disease symptoms appeared at the end of June in the form of the chlorosis on some of the leaves, which became intensely red one week later, while the previously healthy leaves from the same branch becme chlorotic. A few days later, all leaves from the infected leaf branch were intensely red. Infected plants also had slower growth compared to the healthy ones with fewer leaf branches developed. The roots of infected plants were less developed, seared, or gummy with or without brown-colored root hair. The presence of the pathogen was detected by sequencing the 16S rRNA. National Center for Biotechnology Information (NCBI) BLAST analyses of the obtained sequence revealed 100% identity of tested strain with deposited Ca. Phytoplasma solani strains from various countries and hosts, all belonging to the “stolbur” group (16SrXII-A). Identity of 99.74% was found when the tested Serbian strain (MF503627) was compared with the reference stolbur strain STOL11 (AF248959). The oxidative damage of membranes in carrot cells was accompanied by a decrease in the content of photosynthetic pigments. Furthermore, for the determination of specific scavenging properties of the extracts, in vitro antioxidant assay was performed. In phytoplasma-infected carrot leaves, there was a greater reduction in the level of glutathione content (GSH); however; flavonoids and anthocyanidins seem to be responsible for the accompanied increased antioxidative capacity against hydroxyl radical and hydrogen peroxide.

The psyllids Cacopsylla melanoneura and Cacopsylla picta reproduce on apple (Malus × domestica) and transmit the bacterium ‘Candidatus Phytoplasma mali’, the causative agent of apple proliferation. Adult psyllids were collected by the beating-tray method from lower and upper parts of the apple tree canopy in the morning and in the afternoon. There was a trend of catching more emigrant adults of C.melanoneura in the morning and in the lower part of the canopy. For C.melanoneura remigrants, no differences were observed. The findings regarding the distribution of adults were reflected by the number of nymphs collected by wash-down sampling. The density of C.picta was too low for a statistical analysis. The vector monitoring and how it is commonly performed, is suitable for estimating densities of C.melanoneura. Nevertheless, above a certain temperature threshold, prediction of C.melanoneura density might be skewed. No evidence was found that other relatively abundant psyllid species in the orchard, viz. Baeopelma colorata, Cacopsylla breviantennata, Cacopsylla brunneipennis, Cacopsylla pruni and Trioza urticae, were involved in ‘Candidatus Phytoplasma mali’ transmission. The results of our study contribute to an advanced understanding of insect vector behavior and thus have a practical impact for an improved field monitoring.

Candidatus Phytoplasma, the causative agent of yellows disease, inflicts substantial damage on several hundred plant species including perennials and annual plants. The endophytic bacterium Frateuria defendens reduces the symptoms of yellows disease in a number of agricultural crops. One possible mode of action is that the bacterium secretes antimicrobial metabolites. To test this hypothesis, the substances secreted by the endophyte during 10 days of growth in an artificial medium were identified by GC-MS (gas chromatography−mass spectrometry). Synthetic analogues to these substances were then used on periwinkle, a nurse culture plant infected by phytoplasma. Phytoplasma quantities were evaluated by quantitative PCR, and disease symptoms were monitored and recorded. It was found that specific compounds identified by the biochemical analysis caused a significant reduction in both the titer of phytoplasma and the disease symptoms in periwinkle when compared to untreated infected plants. Further research is required to examine the potential of these compounds as an effective treatment against yellows disease.

In Limonium sinuatum Mill, plants with severe symptoms of aster yellows infection phytoplasmas were present not only in the phloem but also in some cortex parenchymas cells. These parenchyma cells were situated at some distance from the conducting bundles. The phytoplasmas were observed directly in parenchyma cells cytoplasm. The number of phytoplasmas present in each selected cell varies. The cells with a small number of phytoplasmas show little pathological changes compared with the unaffected cells of the same zone of the stem as well with the cells of healthy plants. The cells filled with a number of phytoplasmas had their protoplast very much changed. The vacuole was reduced and in the cytoplasm a reduction of the number of ribosomes was noted and regions of homogenous structure appeared. Mitochondria were moved in the direction of the tonoplast and plasma membrane. Compared to the cells unaffected by phytoplasma, the mitochondria were smaller and had an enlarged cristae internal space. The chloroplasts from affected cells had a very significant reduction in size and the tylacoids system had disappeared. The role of these changes for creating phytoplasma friendly enviroment is discused.

Changes in anatomy and cytology of conducting tissues of Limonium sinuatum Mill. plants affected by aster yellows phytoplasma were investigated. In the phloem tissues of affected plants stem necrosis takes place. In necrotic regions no sieve tubes were observed only necrotic cells and parenchyma cells. The sieve tubes present on the border of necrosis showed collapsed walls and were rich in vesicles. Phytoplasma cells were observed in sieve tubes present in nonnecrotic regions of the phloem. Various structural changes in sieve elements were investigated. The endoplasmic reticulum cistemae were often localised in the lumen of the sieve element without contact with the walls. Such localisation of endoplasmic reticulum was never observed in healthy plants. Vesicles of different size, fuzzy material and clumping of p-proteins were characteristic for sieve elements from nonnecrotic part of phloem. No correlation with the sieve tube structure and the appearance of phytoplasma in a single sieve element was found. In control plants of L. sinuatum phloem observed were phloem parenchyma cells with spiny vesicles (SV). In infected plants there were a remarkable increase in cells with SV. Also the SV itself had not only a vesicular but also a tubular or extended cistern shape.

Healthy and AY-affected plants of L.sinuatum have been propagated in vitro for 12 months on the media with and without cytokinins. In the contrary to the healthy plants the phytoplasma affected statice showed abnormal proliferation of the axillary shoots, shortening of the internodes, smaller leaves and severe chlorosis. On the medium without cytokinins, diseased plants proliferated and formed 7.0 axillary shoots per explant but the healthy ones only formed 2.3 shoots; however, the fresh weight of them was similar. On the media with cytokinins, the multiplication rate and fresh weight of healthy shoots greatly in-creased, but of the diseased plants were on the same laevel or decreased. During tissue culture phytoplasma could be detected in symptomatic plants by PCR as well as electron microscope however, the phytoplasmas showed the symptoms of degeneration.

Pleomorphic phytoplasmas were observed in sieve tubes, companion cells and in phloem parenchyma of Tagetes patula L., Helichrysum bracteatum Willd. and Gladiolus sp. L. plants with morphological changes typical for phytoplasma infection. In the pores of the sieve plate phytoplasma cells were seen which suggests that the vertical transport of this pathogen goes in the sieve tubes of infected plants throughout the sieve tube pores. The contact of the sieve tube with the neighbouring cells goes through the plasmodesmata, but no changes of the plasmodesmata were observed in the phloem of infected plants. The size and structure of unchanged plasmodesmata does not allow passing through such big structures like phytoplasma. Instead close contact between phytoplasma cells and vertical sieve tube walls takes place. Damages to the cell wall were observed forming cavities in which the phytoplasma cells were present. The damages of parenchyma and companion cells walls also were seen. In cells where the damages of the walls were observed phytoplasmas were present. The phytoplasma cells were sporadically seen also in the intercellular spaces of parenchyma. These data suggest that horizontal transport depends on damages to the infected plant cell walls caused by the phytoplasma itself.

To test whether phytoplasmas are sensitive to temperature, phytoplasma affected micropropagated gladiolus plants were grown under varying conditions of media content and temperature, in the presence or absence of light. PCR analysis indicated that phytoplasma detection was more successful in plants grown at low temperatures. Plants kept from one to three months at reduced temperature tended to have higher titre of phytoplasma than the plants maintained in stable 20oC high temperature. The best detection was in plants grown on medium containing kinetin+NAA and in the presence of light. In those plants phytoplasmas were detected in direct PCR after one month of culture.

Roots of Gladiolus x hybridus Van Houtte plants infected with aster yellows phytoplasma were examined. The infected plants had a reduced root system in comparison to control plants. Their roots were thinner and the stele organisation was changed. Phytoplasmas were present in sieve tubes, companion cells and phloem parenchyma cells of the infected plant roots. Free calcium ions were localized in the cells of infected plants. Cells of the stele of infected roots, especially these infected with phytoplasmas, showed an increase of calcium antimonite deposits in theirs protoplasts. Also the number of calcium antimonite deposits increased in sieve tubes of infected roots. The deposits were present on plasma membrane, around the sieve tube plate and also in the lumen of the sieve tube. The increase of free calcium ions in sieve tubes did not cause the occlusion of sieve tube pores. Companion cells and some parenchyma cells with phytoplasmas did not react to phytoplasma infection with an increase of Ca2+ ions in protoplast. The parenchyma cells showing signs of degeneration reacted with high increase of calcium ions. The Ca2+ ions were present mainly in cytoplasm of infected parenchyma cells. There were calcium antimonite deposits in infected plant roots xylem elements and in intracellular spaces of cortex parenchyma. Such deposits were not present in control plants.

The results of PCR examination indicated that during two years of tissue culture at standard conditions, on the medium with BAP 1 mg l-1 and continuous temperature of 20oC, phytoplasma could be detected in diseased plants of rose cv Sacha and Jazz. In the second year of micropropagation phytoplasma detection rate in tissues of infected roses increased and was relatively higher than in the first one. To test whether phytoplasmas are sensitive to temperature and light intensity, phytoplasma-affected micropropagated rose plants were grown on medium with BAP 1.0 or 0.5 mg l-1 and at the temperature of 4, 15, 20 or 25oC in darkness or in the light. PCR analysis indicated that phytoplasma detection was not effected by these conditions during 4 weeks of culturing. However, phytoplasma was not detectable in rose plants after 8 weeks culturing on the same medium without transplanting. Micropropagated rose shoots maintained on medium with Gentamycin or Baytril at the concentration of 25.0 or 50.0 mg l-1 had reduced growth and were chlorotic. However, no direct effect of applied antibiotics on phytoplasma detection was evidenced.

Witches’ broom (WB), associated with the presence of ‘Candidatus Phytoplasma aurantifolia’, is one of the most serious diseases of acid lime. This study determined incidence, distribution, and development of the disease, and morphological changes in leaves and branches of affected host plants. Survey in different parts of Oman showed that WB occurs in most regions in the country, where 108 out of 158 (68%) surveyed farms were found to have diseased trees. A survey of 6,926 acid lime trees showed that severity of WB was positively related (r = 0.948; P