Mycoplasma-like Organisms Research Articles

Mycoplasmalike organisms (MLOs) and distinguish them from other MLOs.

DNA of 10 lines of rice yellow dwarf (RYD) mycoplasmalike organisms (MLOs) from Japan, the Phillippines, and Thailand hybridized with four probes containing chromosomal and six probes containing extrachromosomal DNA of a Tochigi (Japan) line of RYD MLO. One chromosomal probe (RYD9) and all six extrachromosomal probes hybridized with various other MLOs (sugarcane white leaf, onion yellows, cineraria witches'-broom, Japanese hornwort witches'-broom, water dropwort wiches'-broom, gentian witches'-broom, udo dwarf, tsuwabuki witches'-broom, pelargonium witches's-broom, peach western-X, and pear decline). DNA from the culturable mollicutes Spiroplasma kunkelii, Spiroplasma citri, Mycoplasma hominis, and Mycoplasma orale did not hybridize with RYD MLO probes. The extrachromosomal DNAs hybridizing with the probes showed variations in electrophoretic behavior.

Revised Taxonomy of the Class Mollicutes: Proposed Elevation of a Monophyletic Cluster of Arthropod-Associated Mollicutes to Ordinal Rank (Entomoplasmatales ord. nov.), with Provision for Familial Rank To Separate Species with Nonhelical Morphology (Entomoplasmataceae fam. nov.) from Helical Species (Spiroplasmataceae), and Emended Descriptions of the Order Mycoplasmatales, Family Mycoplasmataceae

On the basis of recent phylogenetic studies of 47 species within the class Mollicutes and recent molecular and physiologic findings that distinguish a new group of non-sterol-requiring insect and plant mollicutes from Acholeplasma species, we propose a revised taxonomy of the class Mollicutes. Order I (Mycoplasmatales) is retained as described earlier (S. Razin and E. A. Freundt, p. 740-742, in N. R. Krieg and J. G. Holt, ed., Bergey's Manual of Systematic Bacteriology, vol. 1, 1984), with provision for a single family (Mycoplasmataceae) and two genera (Mycoplasma and Ureaplasma) for classification of sterol-requiring mollicutes primarily associated with vertebrates. Emended descriptions of the order and family for the above taxa are hereby included. Proposed order II (Entomoplasmatales ord. nov.) contains subtaxa for either nonhelical (Entomoplasmataceae fam. nov.) or helical (Spiroplasmataceae) mollicutes; the latter family is hereby transferred from the order Mycoplasmatales. The new order and family permit designation and classification of a monophyletic cluster of mollicute strains associated primarily with arthropods. It is proposed that nonhelical, sterolrequiring insect and plant mollicutes previously designated Mycoplasma ellychniae (Tully et al., 1989), Mycoplasma melaleucae (Tully et al., 1990), Mycoplasma somnilux (Williamson et al., 1990), Mycoplasma luminosum (Williamson et al., 1990), and Mycoplasma lucivorax (Williamson et al., 1990) be transferred to Entomoplasma gen. nov., family Entomoplasmataceae fam. nov., as Entomoplasma ellychniae comb. nov., Entomoplasma melaleucae comb. nov., Entomoplasma somnilux comb. nov, Entomoplasma luminosum comb. nov., and Entomoplasma lucivorax comb. nov. Furthermore, the genus Mesoplasma gen. nov. within the family Entomoplasmataceae fam. nov. is proposed for four non-sterol-requiring insect and plant mollicutes. This designation requires the transfer of Acholeplasma florum (McCoy et al., 1984), Acholeplasma entomophilum (Tully et al., 1988), Acholeplasma seiffertii (Bonnet et al., 1991), and Mycoplasma lactucae (Rose et al., 1990) to Mesoplasma florum comb. nov., Mesoplasma entomophilum comb. nov., Mesoplasma seiffertii comb. nov., and Mesoplasma lactucae comb. nov., respectively. Previously established orders Acholeplasmatales and Anaeroplasmatales within the class Mollicutes are designated orders III and IV, respectively, in the proposed revision. Classification and characteristics of lower taxa within these two orders remain as previously given. The proposal outlined here does not consider the large cluster of nonculturable, arthropod-associated, plant-pathogenic mollicutes (“mycoplasma-like organisms”) recently shown to be more closely related to members of the order Acholeplasmatales.

Association of mycoplasma‐like organisms with tomato big bud disease in Greece

The big bud disease of tomato is described in Greece for the first time. Its identity and aetiology were ascertained by electron microscopy, fluorescence and bright‐field microscopy, transmission tests and antibiotic treatments. Pleomorphic mycoplasma‐like organisms (MLOs) were detected in the sieve tubes of infected leaf tissues by thin‐section electron microscopy and also by fluorescence and bright‐field microscopy after staining with the DNA‐binding fluorescent stain Hoechst 33258, and with toluidine blue. Infected tomato plants showed remission of symptoms when treated with tetracycline‐HCl, but not with benzylpenicillin, while healthy plants developed big bud symptoms when graft inoculated with diseased tomato shoots. The occurrence of natural spread of big bud disease in Greece indicates that one or more vector species are present in the country.

Classification of plant-pathogenic mycoplasma-like organisms using restriction-site analysis of PCR-amplified 16S rDNA

Summary: A method has been developed to amplify the 16S rRNA gene of plant-pathogenic mycoplasma-like organisms (MLOs) from infected plant material using the polymerase chain reaction (PCR). The procedure is dependent on the presence of a BclI restriction site in the 16S rDNA of chloroplasts but not in that of the MLOs. This difference permits the specific amplification of the 16S rDNA of the MLOs from BclI-digested total DNA from infected plants using primers from conserved regions of this gene. In this study 16S rDNA was obtained from 52 MLO isolates from herbaceous dicots and monocots as well as woody plants. Digestion of the 16S rRNA genes using AluI endonuclease revealed seven restriction patterns, which were used to group the isolates examined. Group I, which is also characterized by the presence of two KpnI sites, consisted of 31 isolates, most of which are from herbaceous dicots. Isolates assigned to groups II to VI were mostly from woody plants, while the isolates of group VII were from monocots or obtained from a leafhopper. The restriction patterns varied little within groups; however, four group I isolates and one group IV isolate differed slightly from the typical patterns of these groups as a result of a deletion or a slight shift of one restriction site. The groupings uncovered by AluI restriction were also obtained by digesting the 16S rDNA with RsaI endonuclease. However, some atypical patterns were observed within group V isolates. The groups described on the basis of restriction digest data were supported by sequence analysis. With one exception, the 16S rDNA of isolates within the same group exhibited 97·8 to 99·5% homology while those of different groups showed 89·6 to 92·0% homology.

Destructive Plant Diseases Caused by Mycoplasma-like Organisms in Asia

Destructive Plant Diseases Caused by Mycoplasma-like Organisms in Asia

Simulataneous amplification and sequencing of genomic DNA (SAS): sequencing of 16S rRNA genes using total genomic DNA from Butyrivibrio fibrisolvens, and detection and genotyping of nonculturable mycoplasma-like organisms directly from total DNA isolated from infected plants

A protocol for simultaneous amplification and sequencing of genomic DNA (SAS) was developed which combines Sanger's dideoxy chain-terminating sequencing method and Mullis' polymerase chain reaction (PCR) technique into a single step procedure. Sequencing ladders were generated in SAS by the addition of ddNTPs into a PCR amplification mixture. The technique allowed the determination of 16S rRNA gene sequence for the identification of the rumen bacterium Butyrivibrio fibrisolvens directly from genomic DNA, without prior amplification by cloning or PCR. It also allowed the detection and genotyping of plant pathogenic mycoplasma-like organisms (MLOs) from a complex total DNA mixture prepared from infected plants without prior amplification.

Molecular Detection of Diverse Mycoplasmalike Organisms (MLOs) Associated with Grapevine Yellows and Their Classification with Aster Yellows, X-Disease, and Elm Yellows MLOs

Polymerase chain reactions (PCR) and restriction analyses of PCR-amplified DNA were used to detect and differentiate strains of mycoplasmalike organisms (MLOs) associated with grapevine yellows detected in naturally diseased grapevines in the United States and Italy. At least three major groups of grapevine-infecting MLOs were delineated. FDVA1 MLO, discovered in yellows-diseased grapevines in Virginia, and flavescence doree MLO strain FDU from northern Italy were classified with X-disease MLOs; grapevine yellows-associated MLO strains FDG from Germany, CA1, CH1, SAN1, and SAN2 from northern Italy, and FDB and FDR from southern Italy were classified with aster yellows MLOs; and flavescence doree MLO strain FDF from France was classified in the elm yellows MLO group [...]

Isolation and characterization of full‐length chromosomes from non‐culturable plant‐pathogenic Mycoplasma‐like organisms

We describe the isolation and characterization of full-length chromosomes from non-culturable plant-pathogenic, mycoplasma-like organisms (MLOs). MLO chromosomes are circular and their sizes (640 to 1185 kbp) are heterogeneous. Divergence in the range of chromosome sizes is apparent between MLOs in the two major MLO disease groups, and chromosome size polymorphism occurs among some related agents. MLO chromosome sizes overlap those of culturable mycoplasmas; consequently, small genome size alone cannot explain MLO non-culturability. Hybridization with cloned MLO-specific chromosomal and 16S rRNA probes detected two separate chromosomes in some MLO 'type' strains. Large DNA molecules that appear to be MLO megaplasmids were also demonstrated. The ability to characterize full-length chromosomes from virtually any non-culturable prokaryote should greatly facilitate the molecular and genetic analysis of these difficult bacteria.

Differentiation of Strains in the Aster Yellows Mycoplasmalike Organism Strain Cluster by Serological Assay with Monoclonal Antibodies

Monoclonal antibodies (MAbs) raised against the tomato big bud (BB) mycoplasmalike organism (MLO), a member of the aster yellows (AY) MLO strain cluster, were employed in dot immunobinding assays. The MAbs reacted only with strains in the AY MLO cluster, and not with any of several other MLOs not affiliated with the AY MLO strain cluster. However, reactions with MLOs in the AY cluster varied by strain. All MAbs reacted with BB and several MLO strains previously termed «aster yellows,» including NAY (eastern AY), OKAY1 (Oklahoma strain), NJAY (New Jersey strain), and AY27 (Alberta strain); but none of the MAbs reacted with certain other strains of AY MLO, including strains termed SAYS (western AY), OKAYS (Oklahoma strain), NYAY (New York strain), and MNAY (Minnesota strain)

Cluster-Specific Polymerase Chain Reaction Amplification of 16S rDNA Sequences for Detection and Identification of Mycoplasmalike Organisms

Oligonucleotide primers were designed for polymerase chain reaction amplification of 16S rDNA from mycoplasmalike organisms (MLOs) that belong to the aster yellows (AY) MLO strain cluster. Primer pairs designated FOR1, FIR1, F2R1, and F4R1 primed the amplifications of DNA sequences of approximately 1,440, 1,320, 1,300, and 660 bp, respectively, when templates consisted of DNAs extracted from plants infected by MLOs known to be members of the AY cluster. No amplifications were observed when templates consisted of DNA extracted from healthy plants or from plants infected by non-AY MLO cluster strains. The polymerase chain reaction primer pairs enable detection of MLOs associated with several different plant diseases and permit rapid and specific identification of their affiliations with AY MLO

Restriction Fragment Length Polymorphism Analyses and Dot Hybridizations Distinguish Mycoplasmalike Organisms Associated withFlavescence Doreeand Southern European Grapevine Yellows Disease in Italy

Biotinylated cloned DNA probes were employed in dot hybridizations and restriction fragment length polymorphism (RFLP) analyses to compare mycoplasmalike organisms (MLOs) associated with two grapevine yellows diseases (strain FDU of flavescence doree MLO from northern Italy and strain FDB of southern European grapevine yellows MLO from southern Italy) and Italian periwinkle virescence disease (MLO strain G from northern Italy). Results from dot hybridizations using six probes containing cloned DNA of MLO strain FDU, of MLO strain G, or of American aster yellows MLO strain AY1, revealed that FDU and FDB shared some regions of DNA sequence homology with one another as well as with MLO strains G and AY1, but all four MLOs were mutually distinguished [...]

Production of Monospecific Polyclonal Antibodies Against Aster Yellows Mycoplasmalike Organism-Associated Antigen

Proteins of mycoplasmalike organisms (MLO) associated with aster yellows were detected by Western blotting in partially purified preparations from aster yellows-infected plants. Polyclonal antiserum produced against aster yellows (AY) MLO recognized a 23-kDa protein in AY-infected plants that was not found in healthy controls. In addition, this antiserum recognized proteins common to AY-infected and healthy samples. Cross-absorption of the PA with healthy plant extracts did not completely eliminate the nonspecific antibodies. Antibodies specific for AY MLO-associated protein were purified from polyclonal antibodies by trapping them on AY MLO protein obtained by electrophoresis of infected plant extracts and transferred onto nitrocellulose [...]

Use of Polymerase Chain Reaction to Produce Oligonucleotide Probes for Mycoplasmalike Organisms

Specific oligonucleotide probes for detecting apple proliferation and clover phyllody MLOs (mycoplasmalike organisms) in infected tissues were derived from a partial sequence of their 16S ribosomal RNA genes and were tested by dot blot hybridization against amplified DNA from both healthy plants and plants infected by MLOs. Probes were disease specific, with the exception of probes designed for the apple proliferation MLO, which reacted with the DNA extracted from a periwinkle infected by the plum leptonecrosis MLO. The sequenced DNAs were obtained by PCR (polymerase chain reaction) from crude nucleic acid extracts with primers that allowed the amplification of an 865-bp DNA fragment from infected but not from healthy periwinkles [...]

The Beet Leafhopper-Transmitted Virescence Agent Causes Tomato Big Bud Disease in California

Mycoplasmalike organisms (MLOs) are associated with big bud disease of tomatoes (Lycopersicon esculentum) in many parts of the world. In only a few cases, however, have the MLOs associated with the disease been characterized. We used biological and genetic data to establish that the causal agent of tomato big bud (TBB) disease in California is the beet leafhopper-transmitted virescence agent (BLTVA) MLO. Healthy Circulifer tenellus leafhoppers acquired the BLTVA MLO from field-collected, symptomatic tomato plants and transmitted it to healthy tomato plants, which developed typical big bud symptoms. Healthy tomatoes inoculated with the BLTVA type line (FC-83-13) also developed the floral gigantism and virescence characteristic of the disease (.)

Universal Amplification and Analysis of Pathogen 16S rDNA for Classification and Identification of Mycoplasmalike Organisms

Regions representing about 80% of the 16S rDNA sequences of 40 mycoplasmalike organism (MLO) strains from North America, Asia, and Europe were amplified by polymerase chain reaction (PCR) using a primer pair designed on the basis of an MLO 16S rRNA gene. This primer pair detected every MLO examined from infected periwinkle (Catharanthus roseus) and some other plants. No PCR products were obtained in samples containing DNA extracted from healthy plants. The partial 16S rDNA sequences amplified from these various MLOs were compared through restriction fragment length polymorphism (RFLP) analyses [...]

Micropropagation of little leaf diseased eggplants infected with mycoplasma-like organisms

By micropropagation of plant tissues infected with mycoplasma-like organism (MLOs), a group of non-culturable plant pathogens, it is possible to multiply the diseased plants through tissue culture methods. The ‘little leaf diseased’ eggplant tissues infected with MLOs were used to establish a micropropagation system for MLOs within the host tissues under defined controlled conditions. Axillary buds were initially cultured on woody plant medium (WPM) at 25±2°C under continuous light (3 000 lux). Sprouted buds proliferated quickly on modified WPM medium containing 1 mg 1 1 BAP within 20 d. Diseased shoots produced 7-8 shoots per explant with regular subculturing onto fresh medium. Many shoots (15) were produced on a medium containing 1 mg I'1 BAP and 0.1 mg 1' IAA. Developed shoots produced callus within 20 d and roots within 40 d from the base of the shoots. In this way 40-50 shoots showing little leaf symptoms were obtained in a single 100 ml Erlenmeyer flask. The micropropagated plantlets continuously g...

Genetic Relatedness of Mycoplasmalike Organisms Affecting Elm, Alder, and Ash in Europe and North America

DNA from strain ULW of the European elm witches'-broom mycoplasmalike organism (MLO) maintained in periwinkle was enriched by repeated bisbenzimide-CsCl density gradient centrifugation and cloned in Escherichia coli. Three cloned DNA fragments of the ULW MLO were used as probes to hybridize Southern blots of HindIII-digested DNA prepared from MLO-infected elm, alder, and ash trees and from periwinkle plants infected by various MLOs [...]

Genetic Relatedness of Mycoplasmalike Organisms Detected inUlmusspp. in the United States and Italy by Means of DNA Probes and Polymerase Chain Reactions

DNA fragments of an elm yellows (EY) mycoplasmalike organism (MLO) from diseased periwinkle (Catharanthus roseus) were cloned in plasmid vector pSP6 and Escherichia coli strain JM83. DNA probes were prepared by nick translation of EY-specific recombinant plasmids with biotinylated nucleotides. None of the EY probes hybridized with DNA from four representative strains of the aster yellows MLO strain cluster. None of the probes tested at 50 C hybridized with DNA of the MLOs of ash yellows, potato witches'-broom, Canadian peach X, clover proliferation, and beet leafhopper-transmitted virescence diseases, but two tested at 42 C hybridized with DNA of one or another of these MLOs [...]

Production of Monoclonal Antibodies Against a Mycoplasmalike Organism Associated with Sweetpotato Witches' Broom

Five stable hybridoma clones secreting specific monoclonal antibodies against the sweetpotato witches' broom (SPWB)-mycoplasmalike organism (MIO) were produced by employing an immunization scheme for the induction of the immunological tolerance of mice to plant antigens prior to the administration of MIO immunogens. Neonatal BAIB/c mice were injected first with nontarget plant antigens present in an immunogen preparation and were immunized with MIO-enriched antigens prepared by Percoll density-gradient fractionation or by enzyme treatments 6 wk later [...]

Detection and Differentiation of Plant-Pathogenic Mycoplasmalike Organisms Using Polymerase Chain Reaction

Specific oligonucleotide primers were synthesized using sequence data of the 16S rRNA gene from three plant-pathogenic mycoplasmalike organism (MLO) groups (I-III). When used as primers in the polymerase chain reaction (PCR), four sets of primer pairs were identified: 1) mollicute-specific, 2) MLO-specific, 3) MLO-group 1-specific, and 4) MLO-group III-specific. MLO 16S rRNA genes were selectively amplified by PCR from infected plants and vector insects. Using these primers and thermal cycling conditions, this technique was effective for detecting MLO 16S rRNA genes from diseased plants and infected insect vectors and for differentiating MLO 16S rRNA genes from similar rRNA genes of eukaryotic host organelles and other microorganisms associated with healthy vector insects [...]