Mycoplasma-like Organisms Research Articles

Presence of Two Sets of Ribosomal Genes in Phytopathogenic Mollicutes

DNA from 28 strains of phytopathogenic mycoplasmalike organisms that represented five primary taxonomic clusters was digested with restriction endonucleases and hybridized with several ribosomal probes. The results indicate the presence of two sets of ribosomal genes in all strains examined. Restriction maps of the two ribosomal operons for a group of 12 aster yellows mycoplasmalike organisms were constructed.

Use of a Biotinylated DNA Probe for Detection of the Aster Yellows Mycoplasmalike Organism in Dalbulus maidis and Macrosteles fascifrons (Homoptera: Cicadellidae)

A DNA probe was used to detect aster yellows mycoplasmalike organism acquisition by the corn leafhopper, Dalbulus maidis (DeLong & Wolcott), an insect that does not transmit aster yellows mycoplasmalike organism, as well as by Macrosteles fascifrons (Stœl), a vector insect. Results show the effectiveness of the probe for pathogen detection in both the non-vector and the vector insect.

Quantification of DNA from the Aster Yellows Mycoplasmalike Organism in Aster Leafhoppers ( Macrosteles fascifrons Stål) by a Competitive Polymerase Chain Reaction

Summary A rapid and accurate assay was developed for the quantification of the aster yellows plant pathogenic mycoplasmalike organism (MLO). The assay was based on the competitive polymerase chain reaction (PCR) technique using the MLO 23S ribosomal gene and adjacent sequence as target template. Amplification from this region was MLO-specific, and DNA from several different aster yellows MLO strains yielded PCR products. For the internal standard, a PCR product was cloned from Bacillus cereus after amplification with the target primers under reduced annealing stringency. Coamplification of MLO and internal standard DNA provided accurate quantification of 1 to 10 7 copies of MLO target DNA. The assay was used to quantify the aster yellows MLO in aster leafhoppers ( Macrosteles fascifrons Stal) after the insects had fed on MLO-infected plants. Female leafhoppers accumulated MLOs much faster than males during the first 15 days following feeding. From 20 to 39 days after initial exposure, the concentration of MLOs in male leafhoppers remained approximately half that of females. Initially, the difference in MLO concentration between male and female leafhoppers may have been due to a greater amount of feeding, and therefore MLO uptake, by female leafhoppers. Later, the difference may have been related to the larger body weight of female leafhoppers. By 39 days after infection, the MLO population had reached stationary phase in the insect, and MLO DNA detected by the assay comprised 0.8–0.9% of the total DNA content of infected leafhoppers.

Differentiation of mycoplasmalike organisms (MLOs) in European fruit trees by PCR using specific primers derived from the sequence of a chromosomal fragment of the apple proliferation MLO

A 1.8-kb chromosomal DNA fragment of the mycoplasmalike organism (MLO) associated with apple proliferation was sequenced. Three putative open reading frames were observed on this fragment. The protein encoded by open reading frame 2 shows significant homologies with bacterial nitroreductases. From the nucleotide sequence four primer pairs for PCR were chosen to specifically amplify DNA from MLOs associated with European diseases of fruit trees. Primer pairs specific for (i) Malus-affecting MLOs, (ii) Malus- and Prunus-affecting MLOs, and (iii) Malus-, Prunus-, and Pyrus-affecting MLOs were obtained. Restriction enzyme analysis of the amplification products revealed restriction fragment length polymorphisms between Malus-, Prunus, and Pyrus-affecting MLOs as well as between different isolates of the apple proliferation MLO. No amplification with either primer pair could be obtained with DNA from 12 different MLOs experimentally maintained in periwinkle.

Determination of the distribution and multiplication sites of Flavescence Dorée mycoplasma-like organisms in the host plant Vicia faba by ELISA and immunocytochemistry

A mycoplasma-like organism (MLO) is known to be the aetiological agent of Flavescence Dorée, an important grapevine yellows disease which occurs in Europe. Indirect enzyme-linked immunosorbent assay (ELISA) was used to monitor the distribution of the pathogen in the experimental host plant Vicia faba, during the course of infection after inoculation by the leafhopper Euscelidius variegatus. Post-embedding colloidal gold indirect immunolabelling was developed to identify, without ambiguity, the various forms of MLO cells in the different infected parts of the plant, by transmission electron microscopy. Silver enhancement of the gold probe gave accurate histological and cellular localization of MLOs in tissue sections, by light microscopy. Both ELISA and immunolocalization first detected MLO in roots 17 days after inoculation with infectious leafhoppers. Small vesicles and filamentous bodies were identified as MLOs in the cytoplasm of phloem cells, and ELISA indicated the occurrence of a multiplication phase in the roots 17–24 days after inoculation. MLOs then reached the collar and systemically colonized the basal axillary shoot. They preferentially multiplied in the apical area of this growing shoot. MLO bodies were located in mature sieve tubes and in non-functional phloem cells on the periphery of the vascular bundles, and necrotic cells were observed in the phloem tissue. Electron dense and distorted MLOs were identified in these collapsed cells.

A DNA extraction method that allows reliable PCR amplification of MLO DNA from "difficult" plant host species.

Faculty of Science, Northern Territory University, Casuarina, Northern Territory, Australia 0811 Molecular techniques have facilitated the study of plant pathogenic mycoplasma-like organisms (MLOs) in the last few years, and PCR has been used to detect MLOs in infected plant and insect tissue. (1-3~ Where very little is known about MLOs and wide-scale screening of plant host species is required, or quarantine restrictions prevent the propagation of infected plant material, it is essential that a universal DNA extraction be developed that can be applied to a range of plant host species to give good quality DNA suitable for molecular applications such as PCR. Some of these species may contain high levels of polyphenolics, making it difficult to obtain good quality DNA. (4) To study MLOs in Australia, a screening program was initiated in which a range of plant host species with suspected MLO-associated diseases were collected. A DNA extraction method was needed to obtain DNA suitable for use in PCR. A number of techniques have been published for extraction of MLO DNA. (1-3s'6) One of the problems we encountered was that DNA extracted by some of these methods deteriorated with t ime when stored at -20~ Furthermore, DNA extracted from some plants with classical MLO-associated disease symptoms did not give an amplified product using PCR. This posed significant problems for longterm genetic studies using stockpiled DNA as well as severe l imitations for a screening program in which a wide range of plant host species have to be accommodated. The method described here gave DNA that produced consistent results in PCR after storage at -20~ It also allowed the detection of MLO DNA by PCR from plant host species that had symptoms but were consistently negative using DNA extracted by other methods. Our method is based on modified protocols and combines MLO enrichment and maintenance in an osmotically supplemented buffer, with polyvinylpyrrolidone (PVP) to complex polyphenolic compounds (7~ followed by selective precipitation of nucleic acids using ce ty l t r i -methylammonium bromide (CTAB) and sodium chloride. (3~

Giant calyx: a disease of eggplant (Solanum melongena) associated with a mycoplasma‐like organism in Brazil

A disease of eggplant (Solanum melongena) associated with a mycoplasma‐like organism (MLO) was found in the Federal District, Brazil. Symptoms were characterized by teratological changes in the flowering structure, reduction and malformation of leaves, proliferation of lateral buds, and an overall plant stunting. Evidence for the association between MLO infection and disease symptoms was obtained through graft‐transmission experiments and electron microscopy. This is the first record of an eggplant disease associated with MLOs in Brazil.

Viruses Detected in Stone Fruit Germplasm Entering the United States

No. accessions Except stone fruit from Canada and cherry seed from all countries, stone fruit germplasm (Prunus spp.) must be sent to the National Plant Quarantine Center on arrival in the United States. Thus, germplasm can be imported only by the U.S. Dept. of Agriculture and must be held in quarantine for 3 to 4 years while it is tested for pathogens (U.S. Dept. of Agriculture, 1992). Prunus spp. is prohibited because it may be infected with submicroscopic pathogens [i.e., viruses, viroids, mycoplasma-like organisms (MLO), or unknown infectious agents]. Compared with other perennial crops, stone fruit seem to be unusually prone to many virus-like disorders. Nemeth (1986) listed and described 161 diseases of the five stone fruit crops that are associated with submicroscopic pathogens. Of these diseases, 40% have been reported only in North America, but another 43% are not known to occur in the United States. Some of these pathogens, such as plum pox (sharka) and tomato ringspot viruses, have been destructive in Europe and the United States, respectively. Other diseases are obscure, do not appear to have economic importance, or may be caused by strains of wellknown pathogens. My objective was to report results of tests completed during the last 3 years for virus and virus-like pathogens in introduced Prunus germplasm. When the Prunus arrived in quarantine, vegetative accessions were budded onto virus-free rootstocks; and accessions of seed were sown after stratification. Resulting plants were closely monitored for symptoms of disease and subjected to a battery of tests for virus and virus-like pathogens. Tests were conducted to detect all known pathogens, whether or not they were already widespread within the United States. The Animal and Plant Health Inspec-

Restriction Fragment Length Polymorphisms Within the Apple Proliferation Mycoplasmalike Organism

AbstractGenetic homogeneity of the mycoplasmalike organisms (MLOs) associated with apple proliferation (AP) disease was examined by comparing samples collected in south‐western Germany, France, Italy, and Romania by Southern blot analysis. The material examined consisted of two periwinkle‐maintained strains of the AP‐MLO, samples from witches' brooms of 48 symptomatic trees, and root samples from 14 latently infected trees. Six cloned chromosomal DNA fragments of one of the periwinkle‐maintained strains were used as probes. These probes, which were specific for the AP‐MLO and other European fruit MLOs, hybridized with DNA of all samples which indicates that the MLO5 examined were closely related with one another. However, restriction fragment length polymorphism was common bands. Therefore, the AP agent can be regarded as a taxonomic entity. The variability among the isolates seems not to due to their geographical origin but might be related to pathological traits.

Molecular diagnosis of mycoplasma-like organisms (MLOs) in plants

Worldwide, yellows diseases impact plants important in human nutrition, the natural environment, and the culture and commerce of humans. Since the presumed pathogens, mycoplasma-like organisms (MLOs), have not been isolated in pure culture in vitro, their study must proceed by other experimental approaches. In a study of disease affecting grapevines in Europe and North America, polymerase chain reactions (PCR) and restriction analyses of PCR-amplified DNA were used to detect and differentiate strains of MLOs associated with grapevine yellows. MLOs were detected both in naturally diseased grapevines and in experimentally inoculated host plants. The data indicated an unexpected genomic diversity among grapevine-infecting MLOs, and supported their classification with MLOs in the aster yellows, X-disease, and elm yellows groups. The presence of diverse MLOs in grapevines provokes consideration that these MLOs may be present in overlapping geographic ranges and that multiple MLO infections may occur in individual plants, increasing the complexity of grapevine yellows epidemiology and control and the significance of sensitive MLO detection in planting stock and phytosanitary-regulated germplasm.

Studies on Taxonomic Relationships of Mycoplasma–like Organisms by Southern blot Analysis

AbstractChromosomal DNA fragments from the mycoplasma‐like organisms (MLOs) associated with American aster yellows, apple proliferation, clover phyllody, and vaccinium witches' broom were cloned. Several MLO‐specific fragments from each of these four isolates and a sequence from the 16S rRNA gene of an aster yellows MLO were used in Southern blot hybridizations to investigate the taxonomic relationships of 26 pathologically and geographically diverse MLOs. These MLOs were divided into four categories according to the symptoms induced in periwinkle. Genotypically, these isolates represented four groups (16S RFLP groups) of a classification based on restriction fragment length polymorphisms (RFLP) and sequencing data of the 16S rRNA gene. Probes from three isolates of one symptom category hybridized with isolates from all symptom categories. This result indicates that classification of MLOs by symptomatology does often not coincide with genetic relationships. The hybridization results confirmed the findings, of the 16S RFLP classification that most MLOs from herbaceous plants, especially those inducing virescence in periwinkle, are interrelated. These isolates, which were assigned to one 16S RFLP group, could be further differentiated in this study. Itcould be shown that aster yellows, clover phyllody, stolbur, and safflower phyllody and sandal spike are caused by distinct MLOs. The MLOs associated with apple proliferation, vaccinium witches' broom, and witches' broom of lime as well as two isolatesfrom, stone fruits could also be recognized as distinct organisms.

Mycoplasma–like Organisms Associated with a Grapevine Yellows Disease Occurring in Italy

AbstractTransmission electron microscopy of tissues from Vitis vinifera L. plants, naturally infected with a yellows disease similar to flavescence dorée (FD), revealed the presence of pleomorphic mycoplasmalike organisms (MLOs). These occurred in varying concentrations in mature sieve tube elements of leaf veins. Generally the concentration was very low, although large populations of MLOs were found colonizing almost the entire lumen of two cells. The micro–organisms varied considerably in size, shape and electron opacity. Spherical, oval and filamentous forms were recognized. The bodies showed the typical ultrastructural details of other known plant pathogenic MLOs. They were bounded by a smooth trilaminar membrane and contained ribosome–likegranules and DNA–like strands.

Identification and grouping of mycoplasmalike organisms associated with grapevine yellows and clover phyllody diseases based on immunological and molecular analyses.

Immunofluorescent staining, dot blot hybridization, PCR, random amplified polymorphic DNA (RAPD) markers, and restriction fragment length polymorphism wee used to study the genetic relatedness among mycoplasmalike organisms (MLOs) associated with several geographically diverse grapevine yellows diseases (CA1, CH1, SA1, and SA2 from Bologna, Italy; GYU from Udine, Italy; GYR from Rome, Italy; and GYG from Germany). The relationship between these and MLOs associated with clover phyllody diseases in Italy (CPhB and CPhC) and Canada (CPhCa) was also examined. Two monoclonal antibodies reacted with MLOs of GYU-, CPhB-, and CPhC-infected periwinkles. Dot blot hybridization with two cloned GYU DNA fragments, GYD-1 and GYD-2 inserts, showed that both hybridized with DNAs of GYU-, CPhB-, and CPhC-infected periwinkles but not with those of GYR and CPhCa. In addition, GYD-1 insert hybridized with DNAs of CA1, CH1, SA1, SA2, and GYG. Three primer pairs were developed in PCR experiments for this study. By using primer set GYD2P1F and GYD2P1R, a 600-bp DNA fragment was amplified only when DNAs from GYU-, CPhB-, and CPhC-infected plants were used as templates. With the primer pair GYD2P1F and GYD2P2R, a 550-bp DNA fragment was amplified from GYU, CPhB, CPhC, and GYG. The primer pair GYD1P1F and GYD1P2R, on the other hand, could amplify all isolates, although the patterns of PCR products were not identical for all isolates.(ABSTRACT TRUNCATED AT 250 WORDS)

Mycoplasmalike Organisms as Causes of Slow Growth and Decline of Trees and Shrubs

Mycoplasmalike organisms are obligate parasites of plants and insects. In recent years, advances have been made in detecting and identifying them, distinguishing among those associated with various plant diseases, assigning them to groups based upon homology of DNA base sequences, and determining their roles in decline of trees and shrubs. These advances are reviewed with special reference to ash yellows and elm yellows. Known and probable relationships among MLOs associated with various plant diseases are tabulated.

454 PB 323 SELECTION FOR ASTER YELLOWS RESISTANCE IN CARROT

Aster yellows, an insect-vectored disease caused by a mycoplasmalike organism, limits vegetable crop production in the Midwestern U.S.A breeding effort was initiated in 1982 to develop aster yellows resistance in carrot. A synthetic population (AYSYN) composed of 5 open-pollinated varieties and 4 inbreds was assembled in 1982. Inbred lines and hybrids were extracted from AYSYN using a variety of methods. Selection in artificially-infected field sites was carried out from 1982 until 1989. Twenty-three inbreds and 3 hybrids were developed from AYSYN during the selection process. Replicated field experiments were conducted in 1990, 1991, and 1993 to determine the relative aster yellows resistance of these lines and hybrids in comparison with 6 check cultivars. Averaged over years, significant differences were detected for percent aster yellows infection among genotypes. Lines selected for resistance had a mean of 12% infected plant per plot as compared to 24% infection for standard cultivars. Results demonstrate the effectiveness of inbreeding and field selection for aster yellows resistance. This breeding effort represents the first report of aster yellows-resistant carrot germplasm.

PCR detection of MLOs in quick decline‐affected pear trees in Italy

SummaryPolymerase chain reaction (PCR) amplification, using primers derived from the 16S rRNA gene, followed by restriction fragment length polymorphism (RFLP) analysis with Alu I restriction endonuclease was used to detect myc‐oplasma‐like organisms (MLOs) associated with pear decline. MLOs were consistently detected in pear trees that suddenly wilted and died within a few days during summer, as well as in pears of the same orchards with symptoms similar to the slow form of pear decline. In both cases the same RFLP pattern was obtained. Declining pear trees were 5 to 8‐yr‐old cvs Williams, Kaiser and Max Red Bartlett grafted on to Pyrus communis seedling rootstocks. All the orchards affected by quick decline had severe attacks of pear psyllid (Cacopsylla pyri) during the year this study was performed and during the previous year. The results showed the suitability of DNA amplification by the polymerase chain reaction for the detection of pear decline MLOs and established that MLOs can be detected in infected tissues of dead trees.

Detection of mycoplasmalike organisms in declining oaks by polymerase chain reaction

AbstractBy polymerase chain reaction, mycoplasmalike organisms (MLOs) were detected in several declining trees of Quercus robur sampled at two locations of Germany. The MLOs associated with declining oaks were, within the 16S rDNA studied, genetically uniform and closely related to the MLOs associated with European stone fruit yellows and apple proliferation.

Immunosorbent electron microscopy and gold label antibody decoration of MLOs from crude preparations of infected plants and vector insects

A simple method is given for immunotrapping, gold labelling and electron microscopy of mycoplasmalike organisms (MLOs) from crude preparations of infected plants or insects, enabling MLOs to be confidently distinguished from host materials in negative stain preparations. The appearance of the MLOs is described. MLOs were trapped from extracts of infected periwinkle plants (Catharanthus roseus) and infected vector leaf hoppers (Macrosteles quadripunctulatus), using electron microscope grids coated with the F(ab)2 portion of specific rabbit IgGs. The MLOs were then decorated with the intact igG and labelled with goat anti‐rabbit IgG conjugated to gold particles 5 or 15 nm in diameter. Preparations were negatively stained in 0.5% ammonium molybdate or left unstained. The MLOs used were European aster yellows (EAY) from plant and vector, and Australian tomato big bud (TBB) from the plant. Preparation time was 4 h; large numbers of MLOs and MLO fragments could be trapped and identified, and EAY could be confidently distinguished from the serologically unrelated TBB. Morphologically, EAY and TBB were indistinguishable, both presenting spheroidal bodies, tubular forms and apparently budding chains of cocci. Bacilliform virus‐like particles were sometimes associated with EAY MLOs, especially those from the vector.

Amplification of plasmid DNA to detect plant pathogenic mycoplasmalike organisms

SummaryThe polymerase chain reaction (PCR) was employed to develop a specific assay for plant pathogenic mycoplasmalike organisms (MLOs). A cloned fragment of a plasmid from a severe strain of western aster yellows (AY)‐MLO was sequenced to identify oligonucleotide primers for PCR. Amplified DNA fragments of the predicted size were obtained from DNA extracted from plants and insects infected with pear decline MLO, beet leafhopper‐transmitted virescence agent, elm yellows MLO and several AY‐MLO strains. No amplification occurred from healthy leafhopper or plant DNA. The PCR‐based assay was over 500 times more sensitive than a _tilized_tion‐based assay which _tilized a cloned AY plasmid fragment as a probe. With the PCR‐based assay, MLOs could be detected using DNA samples of leafhoppers that were only crushed and boiled in buffer. Amplification of the target DNA was confirmed by digestion of the PCR product with Mbo I which yielded predicted sized fragments for all MLO strains except Bradford AY and eastern AY. Sequencing the PCR product from elm yellows and eastern AY‐MLOs revealed greater than 90% homology, and the failure to restrict the PCR product with Mbo I was due to a single base change in the restriction endonuclease site. The ability of the assay to detect a wide range of MLOs with minimal sample preparation and high sensitivity will be useful in epidemiological studies of MLO‐caused diseases.

Murine chronic tubulointerstitial nephritis: induction by human uveitis mycoplasma-like organisms

Mycoplasma-like organisms (MLO) are non-cultivated intracellular cell wall deficient pathogenic bacteria with a distinctive ultrastructural appearance. Diagnosis of MLO disease rests on finding the organisms in parasitized cells using a transmission electron microscope. MLO are a well studied cause of transmissible chronic plant vascular disease responsive to antibiotics. MLO have recently been found to cause human chronic progressive ophthalmic disease including uveitis. In human uveitis MLO are readily found within parasitized intraocular fluid leucocytes. Inoculation of human uveitis MLO into mouse eyelids produced a high incidence of chronic progressive ophthalmic disease including uveitis. MLO also disseminated to produce randomly distributed lethal systemic inflammatory disease. MLO parasitized leucocytes and microvascular alterations were found in the disease sites by electron microscopy. This report describes the chronic progressive tubulointerstitial nephritis in 12 of 100 human uveitis MLO eyelid inoculated mice versus 0 in 200 control mice (p < 0.0001). MLO parasitized leucocytes accompanied by tubular and microvascular alterations were found by electron microscopy in all 12 inflamed kidneys versus 0 of 4 control kidneys. Pathobiology of the MLO-induced murine nephritis, resemblance of this nephritis to human idiopathic chronic tubulointerstitial nephritis, bacterial molecular biology, and antibiotic therapy of MLO disease are discussed.