Cells of the nonhelical strain of spiroplasma citri underwent changes of morphology comparable to. We describe the first case of human systemic infection caused by spiroplasma bacteria in a patient with hypogammaglobulinemia undergoing treatment with biological diseasemodifying antirheumatic agents. Integrative and free spiroplasma citri oric plasmids. Spiroplasmacells 109 werecollectedbycentrifugation 16h afterinfection, washedin 1. Pdf the abundant extrachromosomal dna content of the. Spiralin is the most abundant protein at the surface of the plant pathogenic mollicute spiroplasma citri and hence might play a role in the interactions of the spiroplasma with its host plant andor its insect vector. The name stubborn should be restricted to the disease in citrus.
Spiroplasma floricola is grown at 34c with the ph kept constant at 7. Cell free extracts were prepared byfour cycles offreezing in liquid 6 e it. Glucose and trehalose pts permeases of spiroplasma citri. Assessment of genetic diversity in spiroplasma citri by rapd, reppcr and bacteriophage primers. Pdf acknowledgments we thank robert deborde and casey crockett of the united states department of agricultureagricultural research service, san joaquin valley agricultural sciences center, parlier, ca for technical assistance. Sequences essential for transmission of spiroplasma citri by. In another medium, ld8a, cs spiroplasma strain i747 grew to a titer of about 2 10 9 cfuml, with a doubling time of 11 12 hr at 31 c. Leafhopers injected with cultured organisms induced typical symptoms of.
Invasion of insect cells by s piroplasma citri involves. Organization and nucleotide sequences of the spiroplasma citri genes for ribosomal protein s2, elongation factor ts, spiralin, phosphofructokinase, pyruvate kinase, and an unidentified protein. Despite the strong similarities between their replication regions, these plasmids coexist in the spiroplasma cells, indicating that they are mutually compatible. Spiroplasma kunkelii is also referred to as corn stunt spiroplasma as it is the causative agent of corn stunt disease, a disease of corn and other. In order to characterize candidate genes potentially involved in spiroplasma transmission and pathogenicity, the genome of s. Speciesspecific and crossreactive monoclonal antibodies to. The spiroplasma heritable bacterial endosymbiont of drosophila. It is a unique approach to measure the absolute copy number of nucleic acid targets without the need of external standards. Droplet digital polymerase chain reaction ddpcr is a method for performing digital pcr that is based on wateroil emulsion droplet technology. Use spiroplasma citri free budwood for all propagations including topworking, though topworking is not advisable because the pathogen moves freely between the scion and rootstock. Spiroplasma turonicum was identified through molecular methods in several blood cultures.
To gain further insight in the interaction between mollicutes and drosophila, we monitored the survival and antimicrobial peptide expression levels of flies after. The phytopathogen spiroplasma phoeniceum was isolated from diseased plants of madagascar periwinkle catharanthus roseus l. A physical and genetic map of the spiroplasma citri genome. Not only their effects on the reproductive manipulation, wolbachia and spiroplasma play defensive roles against broad enemies, such as. Speciesspecific and crossreactive monoclonal antibodies. The impact of citrus stubborn disease, caused by spiroplasma citri, on citrus. Transmission of different isolates of spiroplasma citri to carrot and citrus by circulifer tenellus hemiptera. Quantitative detection of spiroplasma citri by real. This study evaluated the applicability of ddpcr as a quantitative detection tool for the spiroplasma citri, causal agent of. Spiroplasma shares the simple metabolism, parasitic lifestyle, friedegg colony morphology and small genome of other mollicutes, but has a distinctive helical morphology, unlike mycoplasma.
The assembly of 20,000 sequencing reads obtained from shotgun and chromosomespecific libraries of the spiroplasma citri genome yielded 77 chromosomal contigs totaling 1,674 kbp 92% of the 1,820kbp chromosome. Shi j, pagliaccia d, morgan r, qiao y, pan s, vidalakis g, ma w. The results of experiments with spiroplasmas reported here, coupled with the report of the extraction from them of an actinlike protein, suggest that these highly motile organisms possess an actinlike mediated. Spiroplasma definition of spiroplasma by medical dictionary. Complete genome sequence of spiroplasma phoeniceum strain. Spiroplasmas were discovered by a member of the usda molecular plant pathology laboratory, dr. Pmc free article ye f, renaudin j, bove jm, laigret f. Antiactinperoxidase staining of the helical wallfree. Application of droplet digital pcr for quantitative.
Arthropods in relation to plant disease transmission of different isolates of spiroplasma citri to carrot and citrus by circulifer tenellus hemiptera. Characterization, epidemiology and management strategies of. Citrus stubborn disease csd, first identified in california, is a widespread bacterial disease found in most arid citrusproducing regions in the united states and the mediterranean. The type species, spiroplasma citri, is pathogenic for citrus e. The vectors of spiroplasma citri are individually included in eu directive 7793. Wolbachia and spiroplasma are two known vertically transmitted facultative symbionts in drosophila mateos et al. Pdf spiroplasma citri, a plant pathogenic mollicute. Here, we report the nucleotide sequence of the 1,791,576bp circular chromosome and three plasmids of strain p40t. Exceeds the 100cfuml requirement for noncultivable indicator cell culture method mycoplasma hyorhinis. It infects the phloem of the affected plant, causing fruit deformities. Spiroplasma citri is a cultivable species of spiroplasma, this bacterium is vectored by leafhoppers and infects the phloem of a variety of plant species bove et al. Differential expression of spiroplasma citri surface protein.
Spiroplasma citri spirciphotos eppo global database. Our first job is to listen to and observe what our customers need, and meet those needs. A number of genes were subsequently localized on the map by the use of appropriate probes. Ordering information assay number assay description regulatory compliance sample requirements 300200gmp. Citrus stubborn disease csd, first identified in california, is a widespread bacterial disease found in most arid citrusproducing regions in the united states and the mediterranean region. This information serves as a resource for comparative analyses of spiroplasmal adaptations to. Pdf molecular detection of spiroplasma citri associated. Spiral forms have been transiently isolated from tseaffected brain tissues in sp4 growth media designed for isolation of spiroplasma spp. Application of droplet digital pcr for quantitative detection. Spiroplasma spp a plant, arthropod, animal and human. Quantitative data of spiroplasma citri cell culture dna with sp1 and orf1 primers in qpcr and ddpcr assays. Pdf spiroplasma citri, the type species of the genus spiroplasma.
Pdf genetic diversity of spiroplasma citri strains from different. It has a spiral shape and moves in a corkscrew motion. Strains of spiroplasma citri journal of bacteriology. To atcc valued customers, atcc stands ready to support our customers needs during the coronavirus pandemic. Partial chromosome sequence of spiroplasma citri reveals. It encodes one single 16s23s5s rrna operon carle et al. Since then, in tests involving over 100 horseradish plants with and without br symptoms tested in 19801981, s. Identification and genetic characterization of pectobacterium spp. Spiroplasma is a genus of wallless, lowgc, small grampositive bacteria of the internal contractile cytoskeleton, with helical morphology and motility. Further references and information were obtained from experts, citations within the references, as well as from gray literature.
Spiroplasma cells are restricted to the phloem sieve tubes and are transmitted from plant to plant by the leafhopper vector circulifer haematoceps. Spiroplasma citri is a plantpathogenic mollicute phylogenetically related to grampositive bacteria. Isolation of spiroplasma citri membranes and characterization of membrane proteins by twodimensional gel electrophoresis. An antiserum made against sodium dodecyl sulfatedenatured actin from invertebrates and coupled to horseradish peroxidase specifically stains the wall free prokaryotespiroplasma citri. Any of various small spiralshaped bacteria of the genus spiroplasma that lack a cell wall, move by means of a.
Polymerase chain reactionbased detection of spiroplasma. Characterisation of a virus infecting the wallfree. The genus is classified within the class mollicutes. Spiroplasma turonicum was identified through molecular methods in. Nov 21, 2019 acquisition of spiroplasma citri through membranes by homopterous insects.
Spiroplasma citri is a phloemlimited bacterium that causes citrus stubborn disease and vegetative disorders in apiaceae, such as carrot and celery, and in other hosts. Plasmid psci6 from spiroplasma citri gii3 confers insect. Spiroplasma citri, a phytopathogenic mollicute, is the causal agent of the citrus stubborn disease. Expression of the spiroplasma phoeniceum spiralin in spiropla sma citri june 1995 journal of bacteriology 17710. The largest chromosomal contigs were positioned on the physical and genetic maps constructed from pulsedfield gel electrophoresis. Spiralin is not essential for helicity, motility, or. Spiroplasma citri is the causative agent of citrus stubborn disease, a plant disease affecting species in the genus citrus. A r p d transmission of different isolates of spiroplasma. The genome size of the spiroplasma estimated from restriction fragments is close to 1780 kbp, the largest. Expression of the spiroplasma phoeniceum spiralin in spiroplasma citri june 1995 journal of bacteriology 17710.
Media ld8 and ld8a were highly suitable for primary isolation of s. The organism is serologically related to, but not identical with, spiroplasma citri, the causative agent of citrus stubborn disease. Use of pathogenfree bud and grafting materials is an effective control measure in areas where. Spiroplasma citri, the causal agent of citrus stubborn disease, is restricted to the phloem sieve tubes in plants. Acquisition of spiroplasma citri through membranes by homopterous insects. It is an obligate parasite, surviving in citrus or in a variety of other host plants, with no saprophytic phase. Characterization of frur as a putative activator of the. Ulrich schummer, hans gerd schiefer, in molecular and diagnostic procedures in mycoplasmology, 1995. Novel diagnosis for citrus stubborn disease by detection of a spiroplasma citri secreted protein.
Leafhopers injected with cultured organisms induced typical symptoms of the corn stunt disease when. Spiroplasma citri and two as yet unclassified mycoplasmas of the genusspiroplasma, the corn stunt spiroplasma css and suckling mouse cataract agent smca, were compared by determination of the. A bacterium must grow in vitro in cell free cultures to allow full characterization of a suspect pathogen. To study spiralin function, mutants were produced by inactivating the spiralin gene through homologous recombination. Spiroplasma is a genus of mollicutes, a group of small bacteria without cell walls. Spiroplasma host interactions can be classified as commensal, pathogenic or mutualist. Spiralins of spiroplasma citri and spiroplasma melliferum.
Spiroplasma citri primerdesign ltdtm 150 tests genesig advanced kit for general laboratory and research use only quantification of spiroplasma citri genomes. The abundant extrachromosomal dna content of the spiroplasma citri gii33x genome. Davis first author, research plant pathologist, florist and nursery crops laboratory. The psci1 to 6 plasmids encode the membrane proteins known as s. The homogenate was passed, under sterile conditions, through a 450nm. Spiroplasma citri is a bacterium species and the causative agent of citrus stubborn disease. Saglio p, lhospital m, laflfche d, dupont g, bove jm, tully jg, freundt ea, 1973. A spiralingreen fluorescent protein gfp translational.
A shorttailed polyhedral virus ai with a plaque morphology typical of temperate phages has been purified from the wallless prokaryote spiroplasma. Spiroplasma citri is a cell wallless, plant pathogenic bacteria that. Spiroplasma spp a plant, arthropod, animal and human pathogen. Davis, who also coined the term spiroplasma for these unusual diseasecausing microbes. Abstract spiroplasma citri, the type species of the genus spiroplasma spiroplasmataceae, mollicutes, is restricted to the phloem sieve tubes and transmitted by phloem sapfeeding insects, as is characteristic of the phytopathogenic mollicutes. Spiroplasma citri is a bacterium species and the causative agent of citrus stubborn disease its genome has been partially sequenced. Differential expression of spiroplasma citri surface protein genes in the plant and insect hosts mariepierre dubrana1,2, laure beven1,2, nathalie arricaubouvery1,2, sybille duret1,2, stephane claverol3, joel renaudin1,2 and colette saillard1,2. Spiroplasma citri gii3 contains highly related lowcopynumber plasmids psci1 to 6. Apr 11, 1992 pmc free article chevalier c, saillard c, bove jm. The insecttransmissible strain gii3 of spiroplasma citri contains plasmids psci16, five of which psci15 encode adhesinlike proteins and one psci6 encodes protein p32, which has been associated with insect transmissibility. Spiroplasma definition of spiroplasma by the free dictionary. Pdf spiroplasma citri, a phloemlimited pathogen, causes citrus stubborn disease csd.
Spiroplama citri, the causal agent of citrus stubborn disease, is a bacterium of the class mollicutes and is transmitted by phloemfeeding leafhopper vectors. Remove replants infected with stubborn disease and replant with disease free trees. Citrus stubborn severity is associated with spiroplasma citri titer. Spiroplasma citri is a bacterium of the family spiroplasmataceae and is the causative agent of. Citrus is the main economic host of spiroplasma citri. Detection methods include indexing in indicator plants, in vitro culturing in specific media, and serological and molecular amplification methods, e. The morocco strain r8a2, designated as the type strain of s.
Realtime pcr also was developed with detection limits estimated to be between 104 and 104 ng by serial dilution of a recombinant s. Pdf integrative and free spiroplasma citri oric plasmids. Spiroplasma species are organisms that normally colonize plants and insects. Sequences essential for transmission of spiroplasma citri. A rapid and sensitive method to detect spiroplasma citri, the causal agent of citrus stubborn. Cloning and sequencing of the replication origin oric of the spiroplasma citri chromosome and construction of autonomously replicating artificial plasmids. Spiroplasma williamson major reference works wiley. Filtrates of tissue homogenates inoculated into brucella media incubated for 14 days at 35 c resulted in high. The majority of spiroplasmas are found to be commensals of insects, arachnids. World distribution of spiroplasma citri spirci eppo global database. A spiroplasma can be isolated and grown continuously in cell free medium from stunted corn or from drosophila injected with sap expressed from diseased corn.
Euscelis plebejus can be used as a vector of the bacterium to experimentally infect white clover trifolium repens. The california department of food and agriculture and stakeholders manage ctv in commercial groves and urban areas by an eradication program and maintenance of pathogen free budwood sources and citrus propagations in commercial nurseries. Novel diagnosis for citrus stubborn disease by detection. Buk rapid pinnacle qpcrtm mycoplasma detection assay ep 2.
Spiroplasma citri, a phloemlimited pathogen, causes citrus stubborn disease csd and can be transmitted from plant to plant by several species of phloemfeeding leafhoppers. Experimental or natural infections also occur in horseradish, periwinkle, radish, broad bean, carrot, and other plant species. The results of experiments with spiroplasmas reported here, coupled with the report of the extraction from them of an actinlike protein, suggest that these highly. Csd is an important disorder in certain warm and arid citrusgrowing. This wallless bacterium is transmitted from plant to plant by phloemfeeding leafhoppers and is restricted to the sieve tube elements of the phloem tissue. The restriction enzyme scini, with the cutting site 5 gcgc 3 cgcg, can be found in s. Speciesspecific and crossreactive monoclonal antibodies to the plantpathogenic spiroplasmas spiroplasma citri and s. Description top of page a helical, motile, cell wall free procaryote, bounded by a single membrane. First human systemic infection caused by spiroplasma. Project methods citrus tristeza virus ctv and spiroplasma citri are both phloemlimited pathogens of citrus and are transmitted by insect vectors. The spiroplasmas are the only mollicutes showing motility and helical morphology, apparently. New developments in the culture of spiroplasma kunkelii, the corn stunt spiroplasma.
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