Genetic Interrelatedness among Clover Proliferation Mycoplasmalike Organisms (MLOs) and Other MLOs Investigated by Nucleic Acid Hybridization and Restriction Fragment Length Polymorphism Analyses

DNA was isolated from clover proliferation (CP) mycoplasmalike organism (MLO)-diseased periwinkle plants (Catharanthus roseus (L.) G. Don.) and cloned into pSP6 plasmid vectors. CP MLO-specific recombinant DNA clones were biotin labeled and used as probes in dot hybridization and restriction fragment length polymorphism analyses to study the genetic interrelatedness among CP MLO and other MLOs, including potato witches'-broom (PWB) MLO. Results from dot hybridization analyses indicated that both a Maryland strain of aster yellows and a California strain of aster yellows are distantly related to CP MLO. Elm yellows, paulownia witches'-broom, peanut witches'-broom, loofah witches'-broom, and sweet potato witches'-broom may be very distantly related, if at all, to CP MLO. A new Jersey strain of aster yellows MLO, tomato big bud MLO, clover phyllody MLO, beet leafhopper-transmitted virescence MLO, and ash yellows MLO are related to CP MLO, but PWB MLO is the most closely related. Similarity coefficients derived from restriction fragment length polymorphism analyses revealed that PWB and CP MLOs are closely related strains and thus provided direct evidence of their relatedness in contrast to reliance solely on biological characterization.     

Detection of Mycoplasmalike Organism of Paulownia Witches′-Broom with DAPI Fluorescence Microscopy

Paulownia witches’-broom infected by mycoplasmalike organism (MLO) has been developed several cytochemical methods for diagnosis. These methods all based on the special stain reactions or abnormal fluorescence in groups of infected sieve elements as a diseased symptom,. not really on the direct detection of MLO under light microscope. This paper deals with the demonstration of MLO specific white fluorescence after DAPI staining with GMA sections of diseased young stems. Such fluorescence was absent in sections from health plants. The results were confirmed by the ulrrastrueture of MLO and the structure of sieve elements showing from PAS-TBO stained GMA sections. The described method may not only be used in accurate diagnosis of MLO diseased in different plants, but is also worth in the studies of MLO distribution in plants, MLO dynamics in plant resting stage and MLO transmission to support the theoretical basis for protection.     

Preparation of mycoplasma immunogens from plants and a comparison of polyclonal and monoclonal antibodies made against primula yellows MLO-associated antigens

A method is described for obtaining from plants partially purified preparations of mycoplasma-like organisms (MLO) which are suitable for use as immunogens for polyclonal or monoclonal antibody production, and as antigens for directly coating ELISA plates. Using this method a mouse monoclonal antibody to primula yellows MLO was prepared, and its characteristics compared with those of primula yellows polyclonal antibodies from rabbits and also against polyclonal antibodies made to similar preparations of European aster yellows MLO. No serological distinction was obtained between any of the homologous or heterologous combinations of antibody and MLO preparation using ELISA, fluorescence microscopy with FITC-labelled antibodies, or immunoprobes of western blots of partially purified MLO preparations. By contrast, there were no cross-reactions between the primula or aster yellows antibodies or MLO preparations and preparations of clover phyllody or tomato big bud MLOs or their respective polyclonal antibodies. The primula yellows MLO monoclonal and polyclonal antibodies, and also the European aster yellows MLO polyclonal antibodies, all appeared to recognize only a single major antigen of approximate M, = 22 400 daltons. Some possible explanations for the apparent specificity of the polyclinic antisera for a single antigen, and the relevance to MLO preparation procedures are discussed.     

Immunolabeling of grapevine flavescence dorée MLO in salivary glands of Euscelidius variegatus: a light and electron microscopy study

Flavescence dorée (FD), a grapevine yellows disease, is caused by a mycoplasma-like organism (MLO). A colloidal gold indirect immunolabeling technique identified MLO in salivary glands of a vector leafhopper, Euscelidius variegatus. After aldehyde fixation, tissue samples were prepared by cryoultramicrotomy or embedding in acrylic resins. Double fixation with aldehydes and osmium retroxide, followed by embedding in epon, was also performed. Thin or semi-thin serial sections were treated with polyclonal anti-FD-MLO rabbit antibodies, then with gold-conjugated anti-rabbit IgG. Labeling was revealed using the silver enhancement technique for light microscopy. MLO in frozen thin sections of glands were efficiently labeled. Optimal results were obtained with 4% paraformaldehyde-0.1% glutaraldehyde fixation and low-temperature embedding in LR White resin. Both scattered MLO and unusual dense forms of MLO were easily detected with the electron-dense gold probe. This method distinguished MLO from other membrane-limited bodies and provided a good tool for studying infection in large regions of FD-infected tissues by light microscopy.     

Isolation and characterization of the mitochondrion-like organelle from Blastocystis hominis

Blastocystis hominis in an unusual protozoan parasite of the human intestinal tract. Previous studies have described the presence of mitochondrial-like structures despite the anaerobic nature of the organism. In this study, we describe a simple and rapid technique to isolate and characterize mitochondrion-like organelles (MLO) from B. hominis. The parasite was disrupted using glass beads and the MLO were collected and purified using a sucrose gradient. Negative staining and transmission electron microscopy of the isolated organelles showed mitochondrial-like structures. B. hominis cells were stained with rhodamine 123 and MitoLight to show the presence of transmembrane potential of the MLO. DAPI staining of the cells suggested the presence of DNA in the MLO. Though brief reports have been made in literature, this study is the first to describe a technique for the isolation of the MLO from this organism. Using this technique of isolation, major metabolic functions of the organelle, their associated macromolecules and intra-mitochondrial location can be extensively studied. The role of MLO in this anaerobic protozoan can be widely investigated using this protocol.     

The Role of Non-Vectored Soil Transmission as a Primary Source of Infection by Pepper Mild Mottle and Cucumber Mosaic Viruses in Glasshouse-Grown Capsicum in Australia

Polyclonal rabbit antibodies, prepared against partially purified mycoplasmalike-organisms (MLO) from Grapevine Flavescence Dorée-infected plant extracts, were used to detect MLO in situ in excised salivary glands of the experimental vector Euscelidius variegates Kbm by an indirect immunofluorescent staining technique. Glands were examined from the fourth to the tenth week after the start of MLO acquisition. MLO detection was compared using this method, the transmission test and the ELISA technique applied to insects corpses after removal of salivary glands.     

Attachment of the Flavescence doree pathogen (MLO) to leafhopper vectors and other insects

Flavescence doree (FD) is an important yellows disease of grapevine, caused by mycoplasma-like-organism (MLO) and is transmitted in the field by the leafhopper Scaphoideus titanus Ball. It can be transmitted in the laboratory between Vicia faba test plants by the leafhopper, Euscelidius variegatus Kbm. A technique to identify a specific attachment system between the MLO and the leafhopper vectors was developed. In this method, called “Double Dot”, extracts of macerated healthy whole insects or organs applied to a support membrane or cryosections of healthy whole leafhoppers, are incubated with a MLO-enriched extract from FD-infected V. faba or FD-infected E. variegatus. Attached MLO cells were identified by immunolabelling using FD-MLO specific monoclonal antibodies. Attachment of MLO cells was obtained on extracts of healthy S. titanus and E. variegatus and on tissues such as salivary glands, hemolymph and alimentary tract. On cryosections, MLO attachment was obtained on acini IV and V of the salivary glands and on some acini III, on the ventriculus of the alimentary tract, and on the abdomen fat bodies. “Double dot” experiments were done using other insect species, and MLO cells attachment was obtained on most MLO-vector insects but also on insects from a few non-vector species.     

Invaginations in plant cell protoplasts containing mycoplasma-like bodies (MLO)

MLO containing invaginations were found in protoplasts of phloem parenchyma cells in symptomless young leaves ofRibes houghtonianum Jancz. infected with a yellows disease. The invaginations originate between the cell wall and plasmalemma, usually at plasmodesmata, and change apparently into superficial vesicles in the protoplast; they are entirely or partially limited by host plasmalemma. The formations mentioned occur in parenchyma cells which contain normal organelles. Sometimes they are divided by a smooth membrane system enclosing MLO. Besides MLO the invaginations contain in some cases slimy fibrils resembling the P-protein in sieve tubes. The MLO bodies seen in invaginations have usually a diameter of 50–250 nm and their plasmalemma (unit membrane) is identical with the plasmalemma of MLO bodies occurring in sieve tubes. However, only few MLO bodies in invaginations are electron dense, so that they resemble naturally degenerated forms of MLO. Similar MLO containing invaginations were formerly described from some leafhoppers transmitting MLO.     

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 1185kbp) 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.     

Nonradioactive Screening Method for Isolation of Disease-Specific Probes To Diagnose Plant Diseases Caused by Mycoplasmalike Organisms

DNA fragments of tomato big bud (BB) mycoplasmalike organism (MLO) in diseased periwinkle plants (Catharanthus roseus L.) were cloned to pSP6 plasmid vectors and amplified in Escherichia coli JM83. A nonradioactive method was developed and used to screen for MLO-specific recombinants. Cloned DNA probes were prepared by nick translation of the MLO recombinant plasmids by using biotinylated nucleotides. The probes all hybridized with nucleic acid from BB MLO-infected, but not healthy, plants. Results from dot hybridization analyses indicated that several MLOs, e.g., those of Italian tomato big bud, periwinkle little leaf, and clover phyllody, are closely related to BB MLO. The Maryland strain of aster yellows and maize bushy stunt MLOs are also related to BB MLO. Among the remaining MLOs used in this study, Vinca virescence and elm yellows MLOs may be very distantly related, if at all, to BB MLO. Potato witches' broom, clover proliferation, ash yellows, western X, and Canada X MLOs are distantly related to BB MLO. Southern hybridization analyses revealed that BB MLO contains extrachromosomal DNA that shares sequence homologies with extrachromosomal DNAs from aster yellows and periwinkle little leaf MLOs.     

Contribution to the problem of mycoplasma-like organism transmigration in plants

Anatomical-histological investigations of slide series revealed that the secondary vascular formations appearing scattered in callus tissue culture derived from a stem piece containing mycoplasma-like organisms (MLO) (potato witches’ broom agent) have no connection with the vascular tissues of the original stem piece. 77% of reconstituted tobacco plants (Nicotiana glauca Grah.) from these callus cultures were infected with MLO. When diseasedNicotiana glauca scions are grafted on healthy tomato plants as stocks, MLO transmigration into the stocks is realized in all cases ten days after grafting; at that time, the scions and stocks are united only by a zone of a few layers of parenchymatous cells without any indication of vascular tissue union between the two components. In both cases, the MLO transmigration could not be realized by passage through sieve pores in sieve tubes. Our results bring indirect evidence of MLO transmigration through parenchymatous cells and thus support the hypothesis of MLO passage through the plasmodesmata.     

Use of Polyclonal Antibodies to Identify Mycoplasma-like organisms (MLOs) From the Sudan and from Thailand

Rabbit polyclonal antibodies prepared against faba bean phyllody MLO from the Sudan reacted with its homologous antigen and with extracts of Catharanthus roseus experimentally infected with the same or a related MLO from Crotalaria saltiana showing symptoms of phyllody disease, as well as with extracts of naturally MLO-infected C. saltiana growing in the field in the Sudan. The antibodies also reacted positively with extracts of C. roseus experimentally infected with Crotalaria juncea phyllody MLO and soybean phyllody MLO from Thailand. Polyclonal antibodies prepared against an MLO associated with witches' broom disease in C. juncea reacted positively in ELISA tests with homologous antigen extracts from naturally infected C. juncea as well as with extracts from experimentally infected C. roseus and with extracts prepared from Sesamum indicum plants with phyllody symptoms growing in Thailand. There was no reaction between these antibodies and extracts from C. roseus plants infected with the MLOs associated with C. juncea phyllody or with soybean phyllody. No cross reactions were observed among the antigens and antibodies of the two MLO groups by immunoflorescence, ELISA or western blotting. However, the molecular weight of the principal protein antigen, determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting was the same for both types of MLO. Serologically-similar MLOs thus occur in the Sudan and in Thailand, where they are associated with phyllody symptoms in C. saltiana and faba bean and with C. juncea and soybean, respectively. A second, serologically distinct MLO group was also found infecting C. juncea and S. indicum in Thailand but MLOs from this group have not yet been identified in crops from the Sudan.     

Über die Pleomorphie von MLO in Catharanthus roseus (Vinca rosea)

About the pleomorphism of MLO in Catharanthus roseus (Vinca rosea) MLO of the flower greening (virescence)-type of Primula in the phloem of Catharanthus roseus (Vinca rosea) were fixed in two different ways. After prefixation with OsO4 they showed a diminished pleomorphic morphology, most MLO were spherical. With the fixation of KARNOVSKY (1965) and subsequent treatments with OsO4 and uranyl acetate, however, an extremely high pleomorphism was observed. Such pleomorphic MLO in single thin sections are probably the expression of a threedimensional network-like connection. The concentration of MLO in the sieve elements seems to influence the pleomorphism only insignificantly.     

Studies of Polyclonal Antibodies for the Detection of MLOs Associated with Faba Bean (Vicia faba L.) Using Different ELISA Methods and Dot-blot

Polyclonal antibodies were raised in rabbits against MLO associated with faba bean (Vicia. faba L.) phyllody which exists in the Sudan. Two indirect ELISA methods were able to detect the MLO antigens. In the former, the whole antigen was directly coated onto plates, while in the second, only the F(ab')₂, fragments of the IgG were used to coat the ELISA plates. Higher detectable efficiency was obtained when the F(ab')₂ method was used. Moreover the obtainable antiserum was found to exhibit a high degree of specificity through which the MLO associated with faba bean phyllody in the Sudan, are serologically differentiated from other isolates of MLO existing in the Sudan as well as European MLO isolates maintained at Versailles, and Spiroplasma citri, causal agent of Citrus Stubborn Disease. The positive reactions obtained with this antiserum against MLO phyllody naturally existing in the Sudan on Crotalaria saltiana and some Catharanthus roseus demonstrate that these plants are potential reservoirs of the disease in the Sudan. The same antiserum was used in order to distinguish healthy and diseased plant preparations using the membrane ELISA method (dot-blot).     

Molecular Phylogeny and Evolution of the Plant-Specific Seven-Transmembrane MLO Family

Abstract Homologues of barley Mlo encode the only family of seven-transmembrane (TM) proteins in plants. Their topology, subcellular localization, and sequence diversification are reminiscent of those of G-protein coupled receptors (GPCRs) from animals and fungi. We present a computational analysis of MLO family members based on 31 full-size and 3 partial sequences, which originate from several monocot species, the dicot Arabidopsis thaliana, and the moss Ceratodon purpureus. This enabled us to date the origin of the Mlo gene family back at least to the early stages of land plant evolution. The genomic organization of the corresponding genes supports a monophyletic origin of the Mlo gene family. Phylogenetic analysis revealed five clades, of which three contain both monocot and dicot members, while two indicate class-specific diversification. Analysis of the ratio of nonsynonymous-to-synonymous changes in coding sequences provided evidence for functional constraint on the evolution of the DNA sequences and purifying selection, which appears to be reduced in the first extracellular loop of 12 closely related orthologues. The 31 full-size sequences were examined for potential domain-specific intramolecular coevolution. This revealed evidence for concerted evolution of all three cytoplasmic domains with each other and the C-terminal cytoplasmic tail, suggesting interplay of all intracellular domains for MLO function.     

Use of Polyclonal Antibodies to Identify Mycoplasma-like organisms (MLOs) From the Sudan and from Thailand

Rabbit polyclonal antibodies prepared against faba bean phyllody MLO from the Sudan reacted with its homologous antigen and with extracts of Catharanthus roseus experimentally infected with the same or a related MLO from Crotalaria saltiana showing symptoms of phyllody disease, as well as with extracts of naturally MLO-infected C. saltiana growing in the field in the Sudan. The antibodies also reacted positively with extracts of C. roseus experimentally infected with Crotalaria juncea phyllody MLO and soybean phyllody MLO from Thailand. Polyclonal antibodies prepared against an MLO associated with witches' broom disease in C. juncea reacted positively in ELISA tests with homologous antigen extracts from naturally infected C. juncea as well as with extracts from experimentally infected C. roseus and with extracts prepared from Sesamum indicum plants with phyllody symptoms growing in Thailand. There was no reaction between these antibodies and extracts from C. roseus plants infected with the MLOs associated with C. juncea phyllody or with soybean phyllody. No cross reactions were observed among the antigens and antibodies of the two MLO groups by immunoflorescence, ELISA or western blotting. However, the molecular weight of the principal protein antigen, determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting was the same for both types of MLO. Serologically-similar MLOs thus occur in the Sudan and in Thailand, where they are associated with phyllody symptoms in C. saltiana and faba bean and with C. juncea and soybean, respectively. A second, serologically distinct MLO group was also found infecting C. juncea and S. indicum in Thailand but MLOs from this group have not yet been identified in crops from the Sudan.     

Occurrence of Mycoplasma-like Organisms in Parenchyma Cells of Cuscuta odorata (Ruiz et Pav.)

In shoots of the dodder Cuscuta odorata mycoplasma-like organisms (MLO) were observed to occur in nearly all mature sieve elements. Their frequency within the sieve elements varied from a few organisms to high concentrations. In addition, MLO, were found in other cell types. By identifying these cells and investigating the location in the vascular tissue three different types of cells infected with MLO could be distinguished: (1) phloem parenchyma cells, (2) parenchyma cells separating the five vascular bundles of C. odorata and (3) cells in the outer region of the vascular system next to the cells looking like an endodermis. Within the cells of the types 2 and 3, MLO occurred in large numbers and at different morphological structures., Therefore, we assume that the MLO multiply in these cells.     

The Genetic Relationship Between Mycoplasma-like Organisms Causing Diseases in the Sudan and Different Continents Revealed by Polymerase Chain Reaction (PCR) Amplification of the 16S rRNA Gene Followed by Restriction Fragment Length Polymorphism Analysis

The genetic relationship between faba bean (Vicia faba L.) phyllody and other mycoplasma-like organism (MLO) diseases has been studied by amplification of the conserved region of the 16S rRNA gene followed by restriction fragment length polymorphism (RFLP) analysis using Alu I restriction endonuclease. The restriction patterns produced by faba bean phyllody MLO were smilar to that of Crotalaria saltiana phyllody MLO which persists throughout the year in the Sudan. These, and serological results clearly confirmed that C. saltiana is a reservoir of faba bean phyllody MLO in the Sudan. Moreover, restriction patterns have also shown that MLOs of other diseases have the same RFLP fragment pattern as faba bean phyllody MLO, including C. juncea witches'broom (Thailand) and tomato big-bud (Australia), which differs from the other selected MLO diseases (Gladiolus aster yellow, clover phyllody and yellow decline of lavender, aqll from France). Fragment patterns also revealed the existence of genetically diverse MLO strains in the Sudan. Faba bean phyllody may be placed in group III including WX, apricot chlorotic leaf roll, golden flaveswcence dorée of grapevine, plum leptonecrosis of Prunus salciana, peachy yellow leaf roll, sunnhemp phyllody from Thailand, and blueberry witches' broom.     

Ultrastructural Visualization of Glucide Determinants in Membranes of Mycoplasma-like Organisms

Periodic acid-thiocarbohydrazide-silver proteinate (PATAg) reaction for poiysaccharide localization was performed on ultrathin sections of Spurr resin-embedded tissues of Physostegia virginina and Catharanthus roseusknown to be infected by mycoplasma-like organisms (MLO). Electron microscopy of PATAg-treated sections revealed heavy silver deposition on hmiting membranes of all MLO. It was not possible to detect differences m silver deposition within the multi stratified membrane structure because of masking by silver-grain deposition. Silver deposition was not found in the cytoplasmic region of the MLO. The results indicate the presence of glucide determinants only in the limiting membrane of the MLO. The significance of this finding is discussed.     

Transmission of a New Mycoplasma-Like Organism (MLO) from Cuscuta odorata (Ruiz et Pav.) to Herbaceous Plants and Attempts to its Elimination in the Vector

A new MLO-type, originating from a holoparasite plant Cuscuta odorata (Ruiz et Pav.) causing stunting and reduction of flower- and leaf size on Catharanthus roseus (L.) G. Don. (HEINTZ 1986) was transmitted to Apium graveolens L., Plantago major L., Bellis perennis L. and Cirsium japonicum hybrid. The observed symptoms on the test plants probably caused by the MLO have not yet been described in the literature. The symptomatology on these herbaceous plants gives further data in order to classify the MLO as a new one which is named Guscuta latent MLO (Cl-MLO). Attempts to transmit the pathogens by the leafboppers Euscelidius variegates (Kirschbaum) and Euscelis lineolatus (Brullé) failed. It also was impossible to elimmate the MLO completely from Cuscuta odorata by heat treatment and antibiotic application. However, we succeeded in eliminating the pathogens from Catharanthus roseus by heat treatment.