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BackgroundSpiroplasma citri comprises a bacterial complex that cause diseases in citrus, horseradish, carrot, sesame, and also infects a wide array of ornamental and weed species. S. citri is transmitted in a persistent propagative manner by the beet leafhopper, Neoaliturus tenellus in North America and Circulifer haematoceps in the Mediterranean region. Leafhopper transmission and the pathogen's wide host range serve as drivers of genetic diversity. This diversity was examined in silico by comparing the genome sequences of seven S. citri strains from the United States (BR12, CC-2, C5, C189, LB 319, BLH-13, and BLH-MB) collected from different hosts and times with other publicly available spiroplasmas.ResultsPhylogenetic analysis using 16S rRNA sequences from 39 spiroplasmas obtained from NCBI database showed that S. citri strains, along with S. kunkelii and S. phoeniceum, two other plant pathogenic spiroplasmas, formed a monophyletic group. To refine genetic relationships among S. citri strains, phylogenetic analyses with 863 core orthologous sequences were performed. Strains that clustered together were: CC-2 and C5; C189 and R8-A2; BR12, BLH-MB, BLH-13 and LB 319. Strain GII3-3X remained in a separate branch. Sequence rearrangements were observed among S. citri strains, predominantly in the center of the chromosome. One to nine plasmids were identified in the seven S. citri strains analyzed in this study. Plasmids were most abundant in strains isolated from the beet leafhopper, followed by strains from carrot, Chinese cabbage, horseradish, and citrus, respectively. All these S. citri strains contained one plasmid with high similarity to plasmid pSci6 from S. citri strain GII3-3X which is known to confer insect transmissibility. Additionally, 17 to 25 prophage-like elements were identified in these genomes, which may promote rearrangements and contribute to repetitive regions.ConclusionsThe genome of seven S. citri strains were found to contain a single circularized chromosome, ranging from 1.58 Mbp to 1.74 Mbp and 1597-2232 protein-coding genes. These strains possessed a plasmid similar to pSci6 from the GII3-3X strain associated with leafhopper transmission. Prophage sequences found in the S. citri genomes may contribute to the extension of its host range. These findings increase our understanding of S. citri genetic diversity.

Spiroplasma citri, a helical motile, wall-less, and cultivable microorganism of the class Mollicutes, is the agent of the citrus stubborn disease. There is currently a lack of data about the presence of this pathogen in Moroccan citrus orchards. This study aims to validate serological and molecular methods for routine S. citri diagnosis in Moroccan citrus groves. To provide an update on the present status of the outbreak of the pathogen in Moroccan citrus orchards, a survey of S. citri was conducted in the main citrus-growing regions of Morocco. A total of 575 leaf samples were collected from citrus trees with symptoms attributable to S. citri infection. Samples were collected during 2020 and 2021 from 23 citrus orchards. The presence of S. citri was tested in all samples using the double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). Using this method, 57 samples were found to be infected with S. citri, 41 samples had doubtful results, and the remaining samples were negative. To corroborate the results of the DAS-ELISA test, 148 samples were chosen for additional molecular testing using conventional polymerase chain reaction (PCR) and real-time PCR (qPCR) based on specific primer pairs targeting three different genes (putative adhesion-like gene P58, putative adhesion gene P89, and spiralin gene). Using primers that target the putative adhesion-like gene P58, S. citri was detected by conventional and real-time PCR amplification from plant tissue with differing degrees of specificity. The results allowed us to determine the incidence of S. citri in all Moroccan citrus orchards, with a wide range of positive samples varying from 6.5% to 78%, and to show that molecular tests, particularly real-time PCR assays that target the putative adhesion-like gene P58, are the most sensitive for making an accurate diagnosis of S. citri. Indeed, the real-time PCR with P58-targeting primers yielded positive results from all positive and doubtful ELISA samples as well as some negative samples, with an OD value close to 1.5× times healthy samples, thus demonstrating a high sensibility of this technique. [ABSTRACT FROM AUTHOR]

Spiroplasma citri, the causal agent of citrus stubborn disease, is widely distributed in citrus groves and causes heavy losses to the citrus industry in southern Iran. In the present study the genetic diversity of S. citri strains isolated from various hosts and locations in the Fars Province in southern Iran was investigated. The bacterium was cultured from stubborn affected citrus trees, sesame and safflower plants, and leafhopper vector Circulifer haematoceps. Based on PCR-RFLP of the spiralin gene (spi), four groups could be clearly identified among Iranian S. citri isolates. Group 1 was prominent with 71.8% of isolates found in both citrus and non-citrus hosts. Members of groups 2 (18%), and 5 (5.1%) were identified only in citrus, and members of group 6 only in non-citrus hosts. Phylogenetic analyses based on spi gene sequence could not clearly discriminate isolates of groups 1 and 2. In most cases, RAPD-PCR allowed the differentiation of isolates in different clades based on location or host. However there was no correlation between classification based on RFLP of spi gene and RAPD-PCR of whole genome. Finally, the study was completed by the demonstration that Iranian isolates found only in non-citrus hosts (group 6) including insect vectors are capable of infecting key lime when experimentally transmitted to the plant via a dodder bridge. [ABSTRACT FROM AUTHOR]

Spiroplasma citri, a helical motile, wall-less, and cultivable microorganism of the class Mollicutes, is the agent of the citrus stubborn disease. There is currently a lack of data about the presence of this pathogen in Moroccan citrus orchards. This study aims to validate serological and molecular methods for routine S. citri diagnosis in Moroccan citrus groves. To provide an update on the present status of the outbreak of the pathogen in Moroccan citrus orchards, a survey of S. citri was conducted in the main citrus-growing regions of Morocco. A total of 575 leaf samples were collected from citrus trees with symptoms attributable to S. citri infection. Samples were collected during 2020 and 2021 from 23 citrus orchards. The presence of S. citri was tested in all samples using the double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). Using this method, 57 samples were found to be infected with S. citri, 41 samples had doubtful results, and the remaining samples were negative. To corroborate the results of the DAS-ELISA test, 148 samples were chosen for additional molecular testing using conventional polymerase chain reaction (PCR) and real-time PCR (qPCR) based on specific primer pairs targeting three different genes (putative adhesion-like gene P58, putative adhesion gene P89, and spiralin gene). Using primers that target the putative adhesion-like gene P58, S. citri was detected by conventional and real-time PCR amplification from plant tissue with differing degrees of specificity. The results allowed us to determine the incidence of S. citri in all Moroccan citrus orchards, with a wide range of positive samples varying from 6.5% to 78%, and to show that molecular tests, particularly real-time PCR assays that target the putative adhesion-like gene P58, are the most sensitive for making an accurate diagnosis of S. citri. Indeed, the real-time PCR with P58-targeting primers yielded positive results from all positive and doubtful ELISA samples as well as some negative samples, with an OD value close to 1.5× times healthy samples, thus demonstrating a high sensibility of this technique.

Citrus stubborn was initially observed in California in 1915 and was later proven as a graft-transmissible disease in 1942. In the field, diseased citrus trees have compressed and stunted appearances, and yield poor-quality fruits with little market value. The disease is caused by Spiroplasma citri, a phloem-restricted pathogenic mollicute, which belongs to the Spiroplasmataceae family (Mollicutes). S. citri has the largest genome of any Mollicutes investigated, with a genome size of roughly 1780 Kbp. It is a helical, motile mollicute that lacks a cell wall and peptidoglycan. Several quick and sensitive molecular-based and immuno-enzymatic pathogen detection technologies are available. Infected weeds are the primary source of transmission to citrus, with only a minor percentage of transmission from infected citrus to citrus. Several phloem-feeding leafhopper species (Cicadellidae, Hemiptera) support the natural spread of S. citri in a persistent, propagative manner. S. citri-free buds are used in new orchard plantings and bud certification, and indexing initiatives have been launched. Further, a quarantine system for newly introduced types has been implemented to limit citrus stubborn disease (CSD). The present state of knowledge about CSD around the world is summarized in this overview, where recent advances in S. citri detection, characterization, control and eradication were highlighted to prevent or limit disease spread through the adoption of best practices.

The effect of Citrus stubborn disease (CSD), caused by Spiroplasma citri, on chemical composition and antimicrobial activities of the essential oils of (Citrus sinensis (L) Osbeck) was studied. The results showed that the essential oil extracted from both healthy and Citrus fruit infected by the stubborn disease did not have the same physicochemical characteristics, but have identical chemical composition, with octanal, β-phellandrene, d-limonene, 1 r-α pinene, and d-limonene as major compounds. The infected fruits contained new toxic compounds (2-cyclohexen-1-ol, 2-methyl-5-(1-methylethenyl)-cis, decanal, trans-p-mentha-2, 8-dienol, 2-cyclohexen-1-ol, and, 1-methyl-4-[1-methy-4-(1-methyletheyl)]-cis), at low concentrations (<1%). Both essential oils showed very interesting antibacterial activity against Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, and Pseudomonas aeruginosa ATCC 27853. However, they did not exert any antifungal activity against Botrytis cinerea. The minimum inhibitory concentration (MIC) for the bacterial strains, which were sensitive to the essential oil extracted from both infected and uninfected C. sinensis, was of 4 µL and 32 µL/mL, respectively. [ABSTRACT FROM AUTHOR]

The beet leafhopper Circulifer tenellus is an important pest of agricultural crops in the United States, where it transmits beet curly top virus, beet leafhopper-transmitted virescence agent phytoplasma, and Spiroplasma citri to numerous crops, affecting yield and quality. Each of these pathogens have been linked to serious disease outbreaks within Washington State in the past century. To mitigate the risk of disease, growers target the beet leafhopper in their insect pest management programs. Knowledge of pathogen prevalence in beet leafhopper populations could help growers make better management decisions, but timely diagnostics is required. Four new assays were developed for the rapid detection of the beet leafhopper-associated pathogens. These include two assays that detect Beet leafhopper transmitted virescence agent (a PCR and a real-time PCR SYBR green assay), a duplex PCR assay that simultaneously detects beet curly top virus and Spiroplasma citri , and a multiplex real-time PCR assay for the simultaneous detection of all three pathogens. The screening of dilution series generated from plant total nucleic acid extracts with these new assays typically led to detection at levels 10- to 100-fold more sensitive than the conventional PCR assays currently used. These new tools will allow the rapid detection of beet leafhopper-associated pathogens in both plant and insect specimens and will have the potential to be used in diagnostic laboratories seeking to disseminate fast and accurate results to growers for implementation in their insect pest monitoring programs., Competing Interests: The author(s) declare no conflict of interest.

Spiroplasma citri, a plant pathogenic bacterium responsible for citrus stubborn disease in citrus, is listed as a quarantine pest in Europe and in other parts of the world. For this reason, the introduction of this pathogen is prohibited in many countries and post-entry quarantine is required for imported citrus plants. Therefore, reliable and highly sensitive detection methods are required to test plants in post-entry quarantine. This paper presents a study that characterizes the test parameters of a number of PCR protocols for the detection of S. citri. First, during intra-laboratory evaluation, we characterized four PCR methods and selected three of them. Second, those three methods were compared in the framework of an inter-laboratory study involving five laboratories. As a result, the real-time PCR developed by Yokomi et al. (Plant Disease, 92, 253–60, 2008) targeting the putative gene of the adhesin P58 is recommended for routine use in post-entry quarantine. [ABSTRACT FROM AUTHOR]

The Asian citrus psyllid Diaphorina citri Kuwayama is a sap‐sucking hemipteran that serves as a vector for Candidatus Liberibacter asiaticus (CLas), the causal agent of citrus huanglongbing (HLB). Salivary secretions play critical roles in insect–host plant interactions and are responsible for the establishment of insect herbivory. In the present study, we aim to identify the major proteins in D. citri saliva. Saliva proteins are collected from the artificial diet upon D. citri feeding. Collected proteins are concentrated, filtered, digested and then analyzed using liquid chromatography‐tandem spectrometry (MS/MS). The MS/MS data are then analyzed by searching the D. citri genome deposited in the NCBI. Eighty‐nine proteins are identified in D. citri saliva. Eighty‐six of these proteins are from D. citri, whereas three proteins are from Wolbachia endosymbiont. The proteins identified belong to several functional groups, including enzymes, cytoskeletal proteins, sheath proteins, receptor proteins, transporter proteins, chromatin‐, RNA‐ and DNA‐binding proteins, and other proteins. These proteins could be involved in many key functions, including detoxification of plant defencee compounds and suppression of plant defence, as well as facilitation of stylet penetration, sap sucking and digestion. The identification of these proteins is an essential step for the understanding of D. citri–plant interactions. The identification of key enzymes in the saliva of D. citri could lead to a discovery of proper inhibitors that disrupt its feeding, consequently preventing the spread of the HLB pathogen. The saliva secreted by Diaphorina citri is collected from the artificial diet after adults feeding through a stretched parafilm membrane.Using a liquid chromatography‐tandem spectrometry technique with reference to the draft genome sequence for D. citri, 89 proteins are identified in D. citri saliva, with three of them belonging to Wolbachia endosymbiont.Diaphorina citri saliva proteins included enzymes, cytoskeletal proteins, sheath proteins, receptor proteins and transporter proteins, as well as chromatin‐, RNA‐ and DNA‐binding proteins, in addition to 37% uncharacterized proteins. [ABSTRACT FROM AUTHOR]

Vegetative disorders similar to those associated with the presence of ‘Candidatus Liberibacter solanacearum’ (CaLsol) were observed in carrot plants in Kairouan, Tunisia from 2014 to 2016. Symptoms including leaf curling, yellowing, bronze and purplish discoloration, stunting of plants and roots, and proliferation of secondary roots, affected 20 to 40% of the carrots in some plots. In order to determine if these symptoms were associated with the presence of CaLsol, and/or ‘Ca. Phytoplasma spp.’ and/or Spiroplasma citri, real-time PCR analyses were conducted using specific primers for these pathogens. CaLsol was detected for the first time in Tunisia and for the first time its haplotypes D and E were detected co-infecting a carrot plant. Furthermore, three samples of carrot seed produced in Kairouan tested positive for the haplotype D, showing a high percentage (35 to 63%) of viable bacterial cells after treatment with propidium monoazide. However, all the tests were negative for ‘Ca. Phytoplasma’ spp. as well as for S. citri. The results highlight that several CaLsol haplotypes are emerging carrot pathogens in new areas. [ABSTRACT FROM AUTHOR]

Droplet digital polymerase chain reaction (ddPCR) is a method for performing digital PCR that is based on water-oil emulsion droplet technology. It is a unique approach to measure the absolute copy number of nucleic acid targets without the need of external standards. This study evaluated the applicability of ddPCR as a quantitative detection tool for the Spiroplasma citri, causal agent of citrus stubborn disease (CSD) in citrus. Two sets of primers, SP1, based on the spiral in housekeeping gene, and a multicopy prophage gene, SpV1 ORF1, were used to evaluate ddPCR in comparison with real time (quantitative) PCR (qPCR) for S. citri detection in citrus tissues. Standard curve analyses on tenfold dilution series showed that both ddPCR and qPCR exhibited good linearity and efficiency. However, ddPCR had a tenfold greater sensitivity than qPCR and accurately quantified up to one copy of spiralin gene. Receiver operating characteristic analysis indicated that the ddPCR methodology was more robust for diagnosis of CSD and the area under the curve was significantly broader compared to qPCR. Field samples were used to validate ddPCR efficacy and demonstrated that it was equal or better than qPCR to detect S. citri infection in fruit columella due to a higher pathogen titer. The ddPCR assay detected both the S. citri spiralin and the SpV1 ORF1 targets quantitatively with high precision and accuracy compared to qPCR assay. The ddPCR was highly reproducible and repeatable for both the targets and showed higher resilience to PCR inhibitors in citrus tissue extract for the quantification of S. citri compare to qPCR. [ABSTRACT FROM AUTHOR]

Mollicutes, including mycoplasmas and spiroplasmas, have been considered as good representatives of the « minimal cell » concept: these wall-less bacteria are small in size and possess a minimal genome and restricted metabolic capacities. However, the recent discovery of the presence of post-translational modifications unknown so far, such as the targeted processing of membrane proteins of mycoplasma pathogens for human and swine, revealed a part of the hidden complexity of these microorganisms. In this study, we show that in the phytopathogen, insect-vectored Spiroplasma citri GII-3 adhesion-related protein (ScARP) adhesins are post-translationally processed through an ATP-dependent targeted cleavage. The cleavage efficiency could be enhanced in vitro when decreasing the extracellular pH or upon the addition of polyclonal antibodies directed against ScARP repeated units, suggesting that modification of the surface charge and/or ScARP conformational changes could initiate the cleavage. The two major sites for primary cleavage are localized within predicted disordered regions and do not fit any previously reported cleavage motif; in addition, the inhibition profile and the metal ion requirements indicate that this post-translational modification involves at least one non-conventional protease. Such a proteolytic process may play a role in S. citri colonization of cells of the host insect. Furthermore, our work indicates that post-translational cleavage of adhesins represents a common feature to mollicutes colonizing distinct hosts and that processing of surface antigens could represent a way to make the most out of a minimal genome. [ABSTRACT FROM AUTHOR]

The near-minimal bacterium Mesoplasma florum constitutes an attractive model for systems biology and for the development of a simplified cell chassis in synthetic biology. However, the lack of genetic engineering tools for this microorganism has limited our capacity to understand its basic biology and modify its genome. To address this issue, we have evaluated the susceptibility of M. florum to common antibiotics and developed the first generation of artificial plasmids able to replicate in this bacterium. Selected regions of the predicted M. florum chromosomal origin of replication (oriC) were used to create different plasmid versions that were tested for their transformation frequency and stability. Using polyethylene glycolmediated transformation, we observed that plasmids harboring both rpmH-dnaA and dnaA-dnaN intergenic regions, interspaced or not with a copy of the dnaA gene, resulted in a frequency of ∼4.1 ⨰ 106 transformants per viable cell and were stably maintained throughout multiple generations. In contrast, plasmids containing only one M. florum oriC intergenic region or the heterologous oriC region of Mycoplasma capricolum, Mycoplasma mycoides, or Spiroplasma citri failed to produce any detectable transformants. We also developed alternative transformation procedures based on electroporation and conjugation from Escherichia coli, reaching frequencies up to 7.87 ⨰ 106 and 8.44 ⨰ 107 transformants per viable cell, respectively. Finally, we demonstrated the functionality of antibiotic resistance genes active against tetracycline, puromycin, and spectinomycin/streptomycin in M. florum. Taken together, these valuable genetic tools will facilitate efforts toward building an M. florum-based near-minimal cellular chassis for synthetic biology. IMPORTANCE Mesoplasma florum constitutes an attractive model for systems biology and for the development of a simplified cell chassis in synthetic biology. M. florum is closely related to the mycoides cluster of mycoplasmas, which has become a model for whole-genome cloning, genome transplantation, and genome minimization. However, M. florum shows higher growth rates than other Mollicutes, has no known pathogenic potential, and possesses a significantly smaller genome that positions this species among some of the simplest free-living organisms. So far, the lack of genetic engineering tools has limited our capacity to understand the basic biology of M. florum in order to modify its genome. To address this issue, we have evaluated the susceptibility of M. florum to common antibiotics and developed the first artificial plasmids and transformation methods for this bacterium. This represents a strong basis for ongoing genome engineering efforts using this near-minimal microorganism. [ABSTRACT FROM AUTHOR]

Spiroplasma citri is a bacterial pathogen responsible for the economically important citrus stubborn disease. Sesame and citrus seeds serve as hosts for both S. citri and its leafhopper vector Circulifer haematoceps. To evaluate whether sesame could act as a reservoir for citrus-infecting strains or not, the genetic diversity among S. citri strains found in leafhoppers collected in citrus and citrus-free sesame fields was investigated. Among 26 periwinkle plants exposed to the collected C. haematoceps leafhoppers, 12 plants developed typical stubborn symptoms. All symptomatic periwinkles were polymerase chain reaction positive using S. citri-specific primer pairs targeting the spiralin and P89 genes. Phylogenetic trees based on spiralin gene sequence analysis indicated that the novel field-collected strains clustered with those belonging to two formerly defined S. citri groups (groups 6 and 1). In addition, our results strongly suggest that group 1 strains could be transmitted from sesame-infected plants to citrus trees by C. haematoceps, while group 6 strains may not infect citrus trees. [ABSTRACT FROM AUTHOR]

Currently, the main arthropod vectored pathogens associated with carrot and celery crop diseases are ˋ Candidatus Liberibacter solanacearum´, Spiroplasma citri and different phytoplasma species. Mitigation strategies require elucidating whether these pathogens survive in the weeds of these Apiaceae crops, which can act as reservoirs. Weed surveys were conducted in a vegetative cycle (April to October 2012) in the spontaneous vegetation that surrounded crops affected by ˋ Ca. L. solanacearum´, S. citri and/or phytoplasmas. Sixty-three species of 53 genera that belong to 23 botanical families were collected in the main carrot and celery Spanish production area. Species were identified, estimating coverage and abundance, and conserved in herbarium. Samples were analysed by nested-PCR with universal primers for phytoplasmas detection, and were sequenced for identification purposes; by conventional PCR for S. citri and real-time PCR for ˋ Ca. L. solanacearum´. The only detected pathogens were ˋ Ca. Phytoplasma trifolii´ (clover proliferation group 16Sr VI-A) in Amaranthus blitoides and Setaria adhaerens and ˋ Ca. P. solani´ (stolbur group 16Sr XII-A) in Convolvulus arvensis. These pathogens were also sporadically detected in celery or carrot crops. Unexpectedly, neither ˋ Ca. L. solanacearum´ nor S. citri was detected in the weed samples, despite the relatively high prevalence of these pathogens (less than 66 % and 25 %, respectively) in the surveyed plots. This suggests that weeds do not play an epidemiological role as reservoirs in the spread of such organisms in the studied region. The use of pathogen-free seed lots and the control of vectors are crucial for preventing the introduction and spread of these economical important pathogens to new areas. [ABSTRACT FROM AUTHOR]

Spiroplasma citri is the pathogen that causes citrus stubborn disease (CSD). Infection of citrus with S. citri has been shown to cause leaf mottling, reduce fruit yield, and stunt tree growth. Fruit from trees exhibiting symptoms of CSD are misshapen and discolored. The symptoms of CSD are easily confused with nutrient deficiencies or symptoms of citrus greening disease. In this study, young Washington navel oranges ( Citrus sinensis ) were graft-inoculated with budwood originating from trees confirmed to be infected with S. citri . Leaf samples were collected monthly for 10 months for metabolomics and differential gene expression analyses. Significant differences in the concentration of metabolites and expressed genes were observed between control and S. citri- infected trees throughout the experiment. Metabolites and genes associated with important defense and stress pathways, including jasmonic acid signaling, cell wall modification, amino acid biosynthesis, and the production of antioxidant and antimicrobial secondary metabolites, were impacted by S. citri throughout the study, and even prior to symptom development. This work fills a current gap in knowledge surrounding the pathogenicity of S. citri and provides an updated mechanistic explanation for the development of CSD symptoms in S. citri- infected plants.

The Panel on Plant Health performed a pest categorisation of Circulifer tenellus (Ct) and C. haematoceps (Ch) (Hemiptera, Cicadellidae) for the European Union (EU) territory. They are well-defined insect species that can be identified on the basis of external morphology and male genitalia. Ch and Ct are considered to originate from the Old World; Ct is also present in North America and the Caribbean. In the EU, Ch is reported in 11 Member States, mostly in southern or central Europe, and Ct is reported in Spain, France, Italy and Greece. Neither species is harmful by itself, but they are vectors of Spiroplasma citri, the causal agent of, for example, citrus stubborn disease. The major impact of Ct in North America results from the transmission of Beet curly top virus to sugarbeet. Ct also transmits ‘Candidatus Phytoplasma trifolii’ and Ch transmits ‘Ca P. asteris’. There is no transovarial transmission of the pathogens. Ch and Ct are regulated harmful organisms in the EU and listed in Annex II, Part A, Section II, of Council Directive 2000/29/EC together with Spiroplasma citri and with respect to plants of Citrus, Fortunella, Poncirus and their hybrids, other than fruit and seeds, despite the fact that Ct and Ch have a larger host range. Ch and Ct are likely to be disseminated by plants for planting (the eggs are laid into the leaf veins and petioles), they have also been observed to hitch-hike on terrestrial vehicles, and Ct is known for its very high flight capacity. Both species have many hosts, in particular in the Chenopodiaceae, Brassicaceae and Asteraceae. Ecological conditions in the risk assessment area are suitable for the establishment and spread of S. citri, at least where citrus is currently grown.

Background: Spiroplasma citri is a cell wall-less, plant pathogenic bacteria that colonizes two distinct hosts, the leafhopper vector and the host plant. Given the absence of a cell wall, surface proteins including lipoproteins and transmembrane polypeptides are expected to play key roles in spiroplasma/host interactions. Important functions in spiroplasma/insect interactions have been shown for a few surface proteins such as the major lipoprotein spiralin, the transmembrane S. citri adhesion-related proteins (ScARPs) and the sugar transporter subunit Sc76. S. citri efficient transmission from the insect to the plant is expected to rely on its ability to adapt to the different environments and more specifically to regulate the expression of genes encoding surface-exposed proteins. Results: Genes encoding S. citri lipoproteins and ScARPs were investigated for their expression level in axenic medium, in the leafhopper vector Circulifer haematoceps and in the host plant (periwinkle Catharanthus roseus) either insect-infected or graft-inoculated. The vast majority of the lipoprotein genes tested (25/28) differentially responded to the various host environments. Considering their relative expression levels in the different environments, the possible involvement of the targeted genes in spiroplasma host adaptation was discussed. In addition, two S. citri strains differing notably in their ability to express adhesin ScARP2b and pyruvate dehydrogenase E1 component differed in their capacity to multiply in the two hosts, the plant and the leafhopper vector. Conclusions: This study provided us with a list of genes differentially expressed in the different hosts, leading to the identification of factors that are thought to be involved in the process of S. citri host adaptation. The identification of such factors is a key step for further understanding of S. citri pathogenesis. Moreover the present work highlights the high capacity of S. citri in tightly regulating the expression level of a large set of surface protein genes, despite the small size of its genome. [ABSTRACT FROM AUTHOR]

Spiroplasma citri is a cell wall-less bacterium that infects plants. It is transmitted by the leafhopper Circulifer haematoceps , which hosts this bacterium in the haemocel and insect tissues. Bacterial factors involved in spiroplasma colonization of the insect host have been identified, but the immune response of the leafhopper to S. citri infection remains unknown. In this study, we showed that C. haematoceps activates both humoral and cellular immune responses when challenged with bacteria. When infected by S. citri, C. haematoceps displayed a specific immune response, evidenced by activation of phagocytosis and upregulation of a gene encoding the protein hexamerin. S. citri infection also resulted in decreased phenoloxidase-like activity. Inhibition of hexamerin by RNA interference resulted in a significant reduction in phenoloxidase-like activity and increased mortality of infected leafhoppers. Therefore, the gene hexamerin is involved in S. citri control by interfering with insect phenoloxidase activity. [ABSTRACT FROM AUTHOR]

Abstract The Panel on Plant Health performed a pest categorisation of Spiroplasma citri, the causal agent of citrus stubborn disease, horseradish brittle root and carrot purple leaf. S. citri is a well‐defined species of the genus Spiroplasma, a group of helical wall‐less bacteria (Mollicutes) mainly associated with arthropods. Routine molecular detection methods are available and are appropriate for identification. S. citri is not known to occur in most of the EU Member States. Among citrus‐growing countries, only Croatia, Greece, Malta and Portugal do not report S. citri. The disease seems to be widespread only in Cyprus. S. citri is included in Annex II, Part A, Section II of Directive 2000/29/EC. The insect vectors Circulifer haematoceps and C. tenellus are included in the same Directive, Annex II, Part A, Section II. Citrus, the main host plant (as a symptomatic host crop), is listed (a) in Annex III, Part A, (b) in Annex IV, Part A, Section II, (c) in the same annex, Part B, and (d) in Annex V, Parts A and B, Section I. S. citri is disseminated by plants for planting and by seven species of leafhoppers (Cicadellidae), of which only three species, C. tenellus, C. haematoceps and C. opacipennis, are reported in the EU. Besides the rutaceous hosts, more than 33 other plant species from 12 different families can be hosts, even if only some may show symptoms. Ecological conditions in the risk assessment area are suitable for the establishment and spread of S. citri at least where citrus is currently grown. Yield reductions on citrus crops are reported from Cyprus, with reduction in fruit size, weight and quality. Infected planting material could contribute to the dissemination of S. citri, as well as the insect vectors. In areas with a hot, dry climate, the impact on the yield and quality may be high.

Spiroplasma citri, the causal agent of stubborn disease, was isolated from leaves and fruits of diseased citrus plants and cultured on solid and liquid C3G medium. On the basis of mode of transmission, symptomatology, shape on solid medium, staining of infected plant tissues with Dienes’ stain and phase contrast microscopy, the isolated agent was identified as Spiroplasma citri. Identifiction was ensured by ELISA and PCR techniques. A fragment (1053 bp) from the spiralin gene of S. citri was amplified by PCR using two specific primers for the spiralin gene. The nucleic acid hybridization techniques (Southern and dot blots) were used for identification of the spiroplasmal genome using non-radioactive DNA probe specific for spiralin gene region. It was carried out as an alternative sensitive method for rapid detection of the Egyption isolate of S. citri using non-radioactive DNA probe specific for spiralin gene region.

Background: Flavescence dorée (FD) of grapevine is a phloem bacterial disease that threatens European vineyards. The disease is associated with a non-cultivable mollicute, a phytoplasma that is transmitted by the grapevine leafhopper Scaphoideus titanus in a persistent, propagative manner. The specificity of insect transmission is presumably mediated through interactions between the host tissues and phytoplasma surface proteins comprising the so-called variable membrane proteins (Vmps). Plant spiroplasmas and phytoplasmas share the same ecological niches, the phloem sieve elements of host plants and the hemocoel of insect vectors. Unlike phytoplasmas, however, spiroplasmas, and Spiroplasma citri in particular, can be grown in cell-free media and genetically engineered. As a new approach for studying phytoplasmas-insect cell interactions, we sought to mimic phytoplasmas through the construction of recombinant spiroplasmas exhibiting FD phytoplasma Vmps at the cell surface. Results: Here, we report the expression of the FD phytoplasma VmpA in S. citri. Transformation of S. citri with plasmid vectors in which the vmpA coding sequence was under the control of the S citri tuf gene promoter resulted in higher accumulation of VmpA than with the native promoter. Expression of VmpA at the spiroplasma surface was achieved by fusing the vmpA coding sequence to the signal peptide sequence of the S. citri adhesin ScARP3d, as revealed by direct colony immunoblotting and immunogold labelling electron microscopy. Anchoring of VmpA to the spiroplasma membrane was further demonstrated by Triton X-114 protein partitioning and Western immunoblotting. Using the same strategy, the secretion of free, functionally active β-lactamase (used as a model protein) into the culture medium by recombinant spiroplasmas was achieved. Conclusions: Construction of recombinant spiroplasmas harbouring the FD phytoplasma variable membrane protein VmpA at their surface was achieved, which provides a new biological approach for studying interactions of phytoplasma surface proteins with host cells. Likewise, the secretion of functional β-lactamase by recombinant spiroplasmas established the considerable promise of the S. citri expression system for delivering phytoplasma effector proteins into host cells. [ABSTRACT FROM AUTHOR]

The Panel on Plant Health performed a pest categorisation of Circulifer tenellus (Ct) and C. haematoceps (Ch) (Hemiptera, Cicadellidae) for the European Union (EU) territory. They are well-defined insect species that can be identified on the basis of external morphology and male genitalia. Ch and Ct are considered to originate from the Old World; Ct is also present in North America and the Caribbean. In the EU, Ch is reported in 11 Member States, mostly in southern or central Europe, and Ct is reported in Spain, France, Italy and Greece. Neither species is harmful by itself, but they are vectors of Spiroplasma citri, the causal agent of, for example, citrus stubborn disease. The major impact of Ct in North America results from the transmission of Beet curly top virus to sugarbeet. Ct also transmits ' Candidatus Phytoplasma trifolii' and Ch transmits ' Ca P. asteris'. There is no transovarial transmission of the pathogens. Ch and Ct are regulated harmful organisms in the EU and listed in Annex II, Part A, Section II, of Council Directive 2000/29/EC together with Spiroplasma citri and with respect to plants of Citrus Fortunella Poncirus and their hybrids, other than fruit and seeds, despite the fact that Ct and Ch have a larger host range. Ch and Ct are likely to be disseminated by plants for planting (the eggs are laid into the leaf veins and petioles), they have also been observed to hitch-hike on terrestrial vehicles, and Ct is known for its very high flight capacity. Both species have many hosts, in particular in the Chenopodiaceae, Brassicaceae and Asteraceae. Ecological conditions in the risk assessment area are suitable for the establishment and spread of S. citri, at least where citrus is currently grown. [ABSTRACT FROM AUTHOR]

Spiroplasma citri is a phloem-limited bacterium causing citrus stubborn disease (CSD). Isolation and culturing of S. citri is technically demanding and time consuming. S. citri is typically low in titer and unevenly distributed in citrus, making reliable detection challenging. The current preferred detection method is polymerase chain reaction (PCR) assays with primers developed from sequences of S. citri housekeeping genes. Recent genome sequencing of S. citri revealed that the bacterium harbors multiple copies of prophage genes. Therefore, targeting multicopy prophage genes was hypothesized to improve sensitivity of PCR detection. Two primer sets, Php-orf1 and Php-orf3, were developed from conserved prophage sequences in the S. citri genome. These primer sets were used to evaluate detection sensitivity in SYBR Green-based quantitative PCR (qPCR) assays with 18 S. citri in cultures isolated from different hosts and locations. Prophage primer set Php-orf1 increased detection sensitivity by 4.91 and 3.65 cycle threshold (Cq) units compared with housekeeping gene primers for spiralin and P58 putative adhesin gene, respectively. Detection was slightly less sensitive for the Php-orf3 primer set at 3.02 and 1.76 Cq units, respectively, over the same housekeeping gene primers. The prophage primer sets were validated for qPCR detection with field samples from three citrus orchards in California's San Joaquin Valley collected from 2007 to 2013. The data showed that S. citri prophage sequences improved sensitivity for qPCR detection of S. citri-infected trees at least 10-fold and reduced the number of false-negative results. No false-positive samples were detected with any of the primer sets. The enhanced sensitivity resulted from the higher copy number of prophage genes in the S. citri genome and, thus, improved CSD diagnosis from field samples. [ABSTRACT FROM AUTHOR]

The Panel on Plant Health performed a pest categorisation of Spiroplasma citri, the causal agent of citrus stubborn disease, horseradish brittle root and carrot purple leaf. S. citri is a well-defined species of the genus Spiroplasma, a group of helical wall-less bacteria (Mollicutes) mainly associated with arthropods. Routine molecular detection methods are available and are appropriate for identification. S. citri is not known to occur in most of the EU Member States. Among citrus-growing countries, only Croatia, Greece, Malta and Portugal do not report S. citri. The disease seems to be widespread only in Cyprus. S. citri is included in Annex II, Part A, Section II of Directive 2000/29/EC. The insect vectors Circulifer haematoceps and C. tenellus are included in the same Directive, Annex II, Part A, Section II. Citrus, the main host plant (as a symptomatic host crop), is listed (a) in Annex III, Part A, (b) in Annex IV, Part A, Section II, (c) in the same annex, Part B, and (d) in Annex V, Parts A and B, Section I. S. citri is disseminated by plants for planting and by seven species of leafhoppers (Cicadellidae), of which only three species, C. tenellus, C. haematoceps and C. opacipennis, are reported in the EU. Besides the rutaceous hosts, more than 33 other plant species from 12 different families can be hosts, even if only some may show symptoms. Ecological conditions in the risk assessment area are suitable for the establishment and spread of S. citri at least where citrus is currently grown. Yield reductions on citrus crops are reported from Cyprus, with reduction in fruit size, weight and quality. Infected planting material could contribute to the dissemination of S. citri, as well as the insect vectors. In areas with a hot, dry climate, the impact on the yield and quality may be high. [ABSTRACT FROM AUTHOR]

The bio-control potential of arbuscular mycorrhizal fungus Glomus mosseae against two pathogenic microorganisms aster yellows (AY) phytoplasma and Spiroplasma citri has been examined in Madagascar periwinkle ( Catharanthus roseus ). G. mosseae had a positive influence on healthy C. roseus plants and S. citri infection. It provided bioprotection against S. citri pathogen and induced significant degree of resistance to spiroplasma infection. Besides, symptom expression significantly reduced and shoot height, leaves number, root fresh and dry weight increased in spiroplasma-infected plants treated with mycorrhiza fungus. Although, G. mosseae had no positive effect on phytoplasma disease. The root architectures were affected by the phytoplasma pathogen, and the root surface area dramatically decreased in G. mosseae treated AY-infected periwinkles compared with the control. Nitrogen and Phosphorus concentrations notably increased in spiroplasma + G. mosseae compared with control plants. Potassium concentration did not differ significantly in all mycorrhizal treated and untreated infected plants except in G. mosseae treated healthy plants. The spore density and root colonization rate did not vary in both pathogen treatments G. mosseae + spiroplasma and G. mosseae + phytoplasma. To our knowledge, this is the first report showing the bioprotective effect of G. mosseae on S. citri . The possible mechanisms involved in complex interaction between plants, cell wall-less bacteria and arbuscular mycorrhizal fungi (AMF) are discussed and the underlying mechanisms for the functioning of AMF are hypothesized. [ABSTRACT FROM AUTHOR]

Spiroplamas are helical, cell wall-less bacteria belonging to the C lass M ollicutes, a group of microorganisms phylogenetically related to low G+ C, Gram-positive bacteria. Spiroplasma species are all found associated with arthropods and a few, including S piroplasma citri are pathogenic to plant. Thus S . citri has the ability to colonize cells of two very distinct hosts, the plant and the insect vector. While spiroplasmal factors involved in transmission by the leafhopper C irculifer haematoceps have been identified, their specific contribution to invasion of insect cells is poorly understood. In this study we provide evidence that the lipoprotein spiralin plays a major role in the very early step of cell invasion. Confocal laser scanning immunomicroscopy revealed a relocalization of spiralin at the contact zone of adhering spiroplasmas. The implication of a role for spiralin in adhesion to insect cells was further supported by adhesion assays showing that a spiralin-less mutant was impaired in adhesion and that recombinant spiralin triggered adhesion of latex beads. We also showed that cytochalasin D induced changes in the surface-exposed glycoconjugates, as inferred from the lectin binding patterns, and specifically improved adhesion of S . citri wild-type but not of the spiralin-less mutant. These results indicate that cytochalasin D exposes insect cell receptors of spiralin that are masked in untreated cells. In addition, competitive adhesion assays with lectins strongly suggest spiralin to exhibit glycoconjugate binding properties similar to that of the V icia villosa agglutinin ( VVA) lectin. [ABSTRACT FROM AUTHOR]

Citrus stubborn disease (CSD), first identified in California, is a widespread bacterial disease found in most arid citrus-producing regions in the United States and the Mediterranean Region. The disease is caused by Spiroplasma citri, an insect-transmitted and phloem-colonizing bacterium. CSD causes significant tree damage resulting in loss of fruit production and quality. Detection of CSD is challenging due to low and fluctuating titer and sporadic distribution of the pathogen in infected trees. In this study, we report the development of a novel diagnostic method for CSD using an S. citri-secreted protein as the detection marker. Microbial pathogens secrete a variety of proteins during infection that can potentially disperse systemically in infected plants with the vascular flow. Therefore, their distribution may not be restricted to the pathogen infection sites and could be used as a biological marker for infection. Using mass spectrometry analysis, we identified a unique secreted protein from S. citri that is highly expressed in the presence of citrus phloem extract. ScCCPP1, an antibody generated against this protein, was able to distinguish S. citri-infected citrus and periwinkle from healthy plants. In addition, the antiserum could be used to detect CSD using a simple direct tissue print assay without the need for sample processing or specialized lab equipment and may be suitable for field surveys. This study provides proof of a novel concept of using pathogen-secreted protein as a marker for diagnosis of a citrus bacterial disease and can probably be applied to other plant diseases. [ABSTRACT FROM AUTHOR]

The first-cultured and most-studied spiroplasma is Spiroplasma citri, the causal agent of citrus stubborn disease, one of the three plant-pathogenic, sieve-tube-restricted, and leafhopper vector-transmitted mollicutes. In Iranian Fars province, S. citri cultures were obtained from stubborn affected citrus trees, sesame and safflower plants, and from the leafhopper vector Circulifer haematoceps. Spiralin gene sequences from different S. citri isolates were amplified by PCR, cloned, and sequenced. Phylogenetic trees based on spiralin gene sequence showed diversity and indicated the presence of three clusters among the S. citri strains. Comparison of the amino acid sequences of eleven spiralins from Iranian strains and those from the reference S. citri strain GII-3 (241 aa), Palmyre strain (242 aa), Spiroplasma kunkelii (240 aa), and Spiroplasma phoeniceum (237 aa) confirmed the conservation of general features of the protein. However, the spiralin of an S. citri isolate named Shiraz I comprised 346 amino acids and showed a large duplication of the region comprised between two short repeats previously identified in S. citri spiralins. We report in this paper the spiralin diversity in Spiroplasma strains from southern Iran and for the first time a partial internal duplication of the spiralin gene. [ABSTRACT FROM AUTHOR]

Abstract: Freshwater prawn Macrobrachium rosenbergii inoculated with 100 μl novel pathogen spiroplasma strain MR-1008 in logarithmic phase (108 spiroplasmas ml−1) were examined for alkaline phosphatase (AKP) activity, acid phosphatase (ACP) activity, superoxide dismutase (SOD) activity, catalase (CAT) activity, as well as expressions of 7 immune related genes in hepatopancreas after 1–28 d. Hematoxylin-eosin (HE) staining showed obvious pathological features in hepatopancreas connective and epithelial tissue. Enzyme activity analyze showed that hepatopancreas AKP and ACP activity increased markedly (P < 0.05) when inoculated with spiroplasma MR-1008 after 5 d and 10 d, respectively. SOD enzyme activity changed less obviously and slightly increased at 1 day post-inoculation, but CAT activity decreased significantly after 5 d inoculation. The expression levels of lipopolysaccharide and β-1,3-glucan-binding protein (LGBP), peroxinectin (PE), α2-macroglobulin (α2M), AKP, ACP, CAT, and copper/zinc SOD (Cu, Zn-SOD) genes in the hepatopancreas were examined by Real-Time PCR (qRT-PCR) and the results demonstrated that these immune related genes were induced by challenge with spiroplasma MR-1008. The results suggested that the prawn immune responses could be activated or inhibited by spiroplasma MR-1008, and that the hepatopancreas also plays key roles in innate immunity for defense against the pathogen. [Copyright &y& Elsevier]