Sequences essential for transmission of spiroplasma citri by. It is an obligate parasite, surviving in citrus or in a variety of other host plants, with no saprophytic phase. Spiroplasma floricola is grown at 34c with the ph kept constant at 7. The abundant extrachromosomal dna content of the spiroplasma citri gii33x genome. Spiroplasma turonicum was identified through molecular methods in. Spiroplasma definition of spiroplasma by the free dictionary. Pdf molecular detection of spiroplasma citri associated. Partial chromosome sequence of spiroplasma citri reveals. In order to characterize candidate genes potentially involved in spiroplasma transmission and pathogenicity, the genome of s. Despite the strong similarities between their replication regions, these plasmids coexist in the spiroplasma cells, indicating that they are mutually compatible. To gain further insight in the interaction between mollicutes and drosophila, we monitored the survival and antimicrobial peptide expression levels of flies after.
Wolbachia and spiroplasma are two known vertically transmitted facultative symbionts in drosophila mateos et al. Speciesspecific and crossreactive monoclonal antibodies to. Cells of the nonhelical strain of spiroplasma citri underwent changes of morphology comparable to. Expression of the spiroplasma phoeniceum spiralin in spiroplasma citri june 1995 journal of bacteriology 17710. Novel diagnosis for citrus stubborn disease by detection of a spiroplasma citri secreted protein. Spiroplasmas were discovered by a member of the usda molecular plant pathology laboratory, dr. Spiroplasma citri is a bacterium species and the causative agent of citrus stubborn disease. 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. Spiroplasma is a genus of mollicutes, a group of small bacteria without cell walls. A bacterium must grow in vitro in cell free cultures to allow full characterization of a suspect pathogen. 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. Identification and genetic characterization of pectobacterium spp. Realtime pcr also was developed with detection limits estimated to be between 104 and 104 ng by serial dilution of a recombinant s. Nov 21, 2019 acquisition of spiroplasma citri through membranes by homopterous insects.
The spiroplasma heritable bacterial endosymbiont of drosophila. Spiroplasma turonicum was identified through molecular methods in several blood cultures. Arthropods in relation to plant disease transmission of different isolates of spiroplasma citri to carrot and citrus by circulifer tenellus hemiptera. Spiroplasma citri is a cell wallless, plant pathogenic bacteria that. Spiroplasma species are organisms that normally colonize plants and insects. The impact of citrus stubborn disease, caused by spiroplasma citri, on citrus. Saglio p, lhospital m, laflfche d, dupont g, bove jm, tully jg, freundt ea, 1973. The homogenate was passed, under sterile conditions, through a 450nm. A physical and genetic map of the spiroplasma citri genome has been constructed using several restriction enzymes and pulsed field gel electrophoresis. Spiroplasma is a genus of wallless, lowgc, small grampositive bacteria of the internal contractile cytoskeleton, with helical morphology and motility. Invasion of insect cells by s piroplasma citri involves.
Quantitative data of spiroplasma citri cell culture dna with sp1 and orf1 primers in qpcr and ddpcr assays. 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. Spiroplasma cells are restricted to the phloem sieve tubes and are transmitted from plant to plant by the leafhopper vector circulifer haematoceps. Remove replants infected with stubborn disease and replant with disease free trees. Apr 11, 1992 pmc free article chevalier c, saillard c, bove jm. 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.
Spiroplasma host interactions can be classified as commensal, pathogenic or mutualist. Transmission of different isolates of spiroplasma citri to carrot and citrus by circulifer tenellus hemiptera. 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. Spiroplasma citri gii3 contains highly related lowcopynumber plasmids psci1 to 6.
Spiroplasma citri is the causative agent of citrus stubborn disease, a plant disease affecting species in the genus citrus. Pmc free article ye f, renaudin j, bove jm, laigret f. Leafhopers injected with cultured organisms induced typical symptoms of the corn stunt disease when. Filtrates of tissue homogenates inoculated into brucella media incubated for 14 days at 35 c resulted in high. Genomic dna from spiroplasma citri strain moroccor8a2, ncppb 2647 typestraintrue application.
Spiralins of spiroplasma citri and spiroplasma melliferum. Spiralin is not essential for helicity, motility, or. A shorttailed polyhedral virus ai with a plaque morphology typical of temperate phages has been purified from the wallless prokaryote spiroplasma. A spiralingreen fluorescent protein gfp translational. Spiroplasma citri is a bacterium of the family spiroplasmataceae and is the causative agent of. Pdf spiroplasma citri, a plant pathogenic mollicute. Citrus is the main economic host of spiroplasma citri. A number of genes were subsequently localized on the map by the use of appropriate probes. Glucose and trehalose pts permeases of spiroplasma citri.
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. Spiral forms have been transiently isolated from tseaffected brain tissues in sp4 growth media designed for isolation of spiroplasma spp. Expression of the spiroplasma phoeniceum spiralin in spiropla sma citri june 1995 journal of bacteriology 17710. Cell free extracts were prepared byfour cycles offreezing in liquid 6 e it. It infects the phloem of the affected plant, causing fruit deformities. Spiroplama citri, the causal agent of citrus stubborn disease, is a bacterium of the class mollicutes and is transmitted by phloemfeeding leafhopper vectors. This study evaluated the applicability of ddpcr as a quantitative detection tool for the spiroplasma citri, causal agent of. Csd is an important disorder in certain warm and arid citrusgrowing. Ordering information assay number assay description regulatory compliance sample requirements 300200gmp. Differential expression of spiroplasma citri surface protein. Spiroplasma citri, a phloemlimited pathogen, causes citrus stubborn disease csd and can be transmitted from plant to plant by several species of phloemfeeding leafhoppers. Pdf spiroplasma citri, a phloemlimited pathogen, causes citrus stubborn disease csd. 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. Spiroplasma definition of spiroplasma by medical dictionary.
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. Organization and nucleotide sequences of the spiroplasma citri genes for ribosomal protein s2, elongation factor ts, spiralin, phosphofructokinase, pyruvate kinase, and an unidentified protein. An antiserum made against sodium dodecyl sulfatedenatured actin from invertebrates and coupled to horseradish peroxidase specifically stains the wall free prokaryotespiroplasma citri. Quantitative detection of spiroplasma citri by real. First human systemic infection caused by spiroplasma. Spiroplasma citri primerdesign ltdtm 150 tests genesig advanced kit for general laboratory and research use only quantification of spiroplasma citri genomes. Assessment of genetic diversity in spiroplasma citri by rapd, reppcr and bacteriophage primers. 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. Droplet digital polymerase chain reaction ddpcr is a method for performing digital pcr that is based on wateroil emulsion droplet technology.
Speciesspecific and crossreactive monoclonal antibodies. The phytopathogen spiroplasma phoeniceum was isolated from diseased plants of madagascar periwinkle catharanthus roseus l. To atcc valued customers, atcc stands ready to support our customers needs during the coronavirus pandemic. It is a unique approach to measure the absolute copy number of nucleic acid targets without the need of external standards. Spiroplasma spp a plant, arthropod, animal and human. Media ld8 and ld8a were highly suitable for primary isolation of s. Spiroplasmacells 109 werecollectedbycentrifugation 16h afterinfection, washedin 1. Plasmid psci6 from spiroplasma citri gii3 confers insect. Application of droplet digital pcr for quantitative. New developments in the culture of spiroplasma kunkelii, the corn stunt spiroplasma. Polymerase chain reactionbased detection of spiroplasma. Project methods citrus tristeza virus ctv and spiroplasma citri are both phloemlimited pathogens of citrus and are transmitted by insect vectors.
Complete genome sequence of spiroplasma phoeniceum strain. Antiactinperoxidase staining of the helical wallfree. Exceeds the 100cfuml requirement for noncultivable indicator cell culture method mycoplasma hyorhinis. Buk rapid pinnacle qpcrtm mycoplasma detection assay ep 2. The organism is serologically related to, but not identical with, spiroplasma citri, the causative agent of citrus stubborn disease. Pdf genetic diversity of spiroplasma citri strains from different. Citrus stubborn severity is associated with spiroplasma citri titer. Spiroplasma citri is a plantpathogenic mollicute phylogenetically related to grampositive bacteria. Davis first author, research plant pathologist, florist and nursery crops laboratory.
Any of various small spiralshaped bacteria of the genus spiroplasma that lack a cell wall, move by means of a. Leafhopers injected with cultured organisms induced typical symptoms of. The type species, spiroplasma citri, is pathogenic for citrus e. Sequences essential for transmission of spiroplasma citri. 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. Spiroplasma citri spirciphotos eppo global database. A physical and genetic map of the spiroplasma citri genome.
The largest chromosomal contigs were positioned on the physical and genetic maps constructed from pulsedfield gel electrophoresis. Here, we report the nucleotide sequence of the 1,791,576bp circular chromosome and three plasmids of strain p40t. Not only their effects on the reproductive manipulation, wolbachia and spiroplasma play defensive roles against broad enemies, such as. Davis, who also coined the term spiroplasma for these unusual diseasecausing microbes. Further references and information were obtained from experts, citations within the references, as well as from gray literature. Euscelis plebejus can be used as a vector of the bacterium to experimentally infect white clover trifolium repens. Integrative and free spiroplasma citri oric plasmids. Characterisation of a virus infecting the wallfree. 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.
This wallless bacterium is transmitted from plant to plant by phloemfeeding leafhoppers and is restricted to the sieve tube elements of the phloem tissue. Shi j, pagliaccia d, morgan r, qiao y, pan s, vidalakis g, ma w. 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. The psci1 to 6 plasmids encode the membrane proteins known as s. Pdf integrative and free spiroplasma citri oric plasmids. 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.
Speciesspecific and crossreactive monoclonal antibodies to the plantpathogenic spiroplasmas spiroplasma citri and s. The majority of spiroplasmas are found to be commensals of insects, arachnids. The genus is classified within the class mollicutes. Novel diagnosis for citrus stubborn disease by detection. 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.
The spiroplasmas are the only mollicutes showing motility and helical morphology, apparently. Ulrich schummer, hans gerd schiefer, in molecular and diagnostic procedures in mycoplasmology, 1995. Strains of spiroplasma citri journal of bacteriology. The genome size of the spiroplasma estimated from restriction fragments is close to 1780 kbp, the largest. 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. This information serves as a resource for comparative analyses of spiroplasmal adaptations to. Use of pathogenfree bud and grafting materials is an effective control measure in areas where. It has a spiral shape and moves in a corkscrew motion.
Spiroplasma williamson major reference works wiley. To study spiralin function, mutants were produced by inactivating the spiralin gene through homologous recombination. Isolation of spiroplasma citri membranes and characterization of membrane proteins by twodimensional gel electrophoresis. Spiroplasma citri, the causal agent of citrus stubborn disease, is restricted to the phloem sieve tubes in plants. Experimental or natural infections also occur in horseradish, periwinkle, radish, broad bean, carrot, and other plant species. 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 vectors of spiroplasma citri are individually included in eu directive 7793. Our first job is to listen to and observe what our customers need, and meet those needs. Pdf the abundant extrachromosomal dna content of the. Pdf spiroplasma citri, the type species of the genus spiroplasma. The name stubborn should be restricted to the disease in citrus. Description top of page a helical, motile, cell wall free procaryote, bounded by a single membrane.
Cloning and sequencing of the replication origin oric of the spiroplasma citri chromosome and construction of autonomously replicating artificial plasmids. Spiroplasma spp a plant, arthropod, animal and human pathogen. A rapid and sensitive method to detect spiroplasma citri, the causal agent of citrus stubborn. 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. Characterization, epidemiology and management strategies of. It encodes one single 16s23s5s rrna operon carle et al. Acquisition of spiroplasma citri through membranes by homopterous insects. A r p d transmission of different isolates of spiroplasma. The restriction enzyme scini, with the cutting site 5 gcgc 3 cgcg, can be found in s.
Detection methods include indexing in indicator plants, in vitro culturing in specific media, and serological and molecular amplification methods, e. Application of droplet digital pcr for quantitative detection. 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. Spiroplasma citri, a phytopathogenic mollicute, is the causal agent of the citrus stubborn disease. World distribution of spiroplasma citri spirci eppo global database.