Mycoplasma-like Organisms in Phloem Elements of Ammobium alatum

Using transmission electron and fluorescent microscopes, Mycoplasma-like organisms (MLOs) were found in phloem cells of stems and leaves of Ammobium alatum. The diameter of these organisms ranged from 0.12–0.73 μm and averaged 0.4 μm. Symptoms induced by MLOs included chlorosis and reddening of leaves and winged stems, plant stunting and flower phyllody. This is the first report of MLOs associated with disease symptoms in A. alatum.     

Mycoplasma-like Organisms Associated with Proliferation and Big Bud of Solanum lycocarpum St. Hil.

Mycoplasma-like organisms (MLOs) were found in sieve tubes of Solanum lycocarpum affected with prolferation and big bud. The MLOs were graft-transmitted to tomato plants which developed similar symptoms. The association of MLOs with affected but not with symptomless S. lycocarpum and tomato suggests that the abnormality is induced by MLOs. The possible role of S. lycocarpum as a reservoir of a strain of the tomato big bud is discussed.     

Correlation between Light and Electron Microscopic Observations and Identification of Mycoplasmalike Organisms using Consecutive 350 nm Thick Sections

Serial sections of 350 nm thickness were used to make a correlation between electron and light microscopic observations. While thionin-acridine orange staining gave a positive result to detect abnormal sieve tubes of phyllody affected Phlox drummondii Hook, when observed under light microscope, the same cells revealed the presence of typical mycoplasmalike organisms (MLOs) in electron microscopic examination. Advantage of 350 nm thick sections in electron microscopy, and the utility of the technique in MLO detection have been discussed.     

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

Chromosomal 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.     

16S rRNA sequence indicates that plant-pathogenic mycoplasmalike organisms are evolutionarily distinct from animal mycoplasmas

The plant-pathogenic mycoplasmalike organisms (MLOs) are so named because they lack cell walls. Many features that are essential to a definitive classification remain uncharacterized, because these organisms have resisted attempts at in vitro culturing. To establish the taxonomic position of the MLOs, the DNA region containing the 16S rRNA gene from a representative of the MLOs has been cloned and sequenced. Sequence comparisons indicate that the MLOs are related to Mycoplasma capricolum and that these two bacteria share their phylogenetic origin with Bacillus subtilis. The low G + C content of this gene and features of its deduced secondary structure further support this grouping. However, the presence of a single tRNAIle gene in the spacer between the 16S rRNA and 23S rRNA genes of the MLOs differentiates the MLOs from other representatives of the mycoplasmas, which indicates an early divergence in the evolution of the members of the class Mollicutes. The presence of certain characteristic oligonucleotides in the 16S rRNA sequence indicates that MLOs may be closely related to acholeplasmas.     

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

Polymerase 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.     

Mycoplasmalike Organisms (MLOs) Associated With the Witches' Broom Disease of Poplar

The witches' broom disease has been recently observed on poplars in Paris and its suburbs. The incidence of the disease appeared to be considerably high along main roads. The electron microscopic examination of 350 nm thick sieve tube sections revealed the presence of wall-less mycoplasmalike organisms (MLOs) in diseased samples of Populus alba var. nivea Wesm. They could not be found in their healthy counterparts.     

Identification and analysis of a genomic strain cluster of mycoplasmalike organisms associated with Canadian peach (eastern) X disease, western X disease, and clover yellow edge.

Genetic interrelatedness among 13 strains of mycoplasmalike organisms (MLOs) from various sources was evaluated by dot hybridization and restriction fragment length polymorphism analyses using cloned DNA probes derived from Canadian peach X (CX) and western X (WX) MLOs. Dot hybridization analysis indicated that CX, WX, and clover yellow edge MLOs are closely related and form a distinct strain cluster that is only distantly related to the 10 other MLOs. Similarity coefficients derived from restriction fragment length polymorphism analysis revealed that CX, WX, and clover yellow edge MLOs represent three distinct genomic types.     

Studies of the mycoplasma-like organism (MLO) in spinach leaves affected by the aster yellows disease

Summary Mycoplasma-like organisms (MLO) were found in the phloem of spinach (Spinacia oleracea L.) leaves infected with the agent of aster yellows disease by means of the leafhopperMacrosteles fascifrons Stål. The MLOs occurred mainly in mature sieve elements but were recorded in occasional phloem parenchyma cells as well. The MLO showed the typical features of this organism. The majority were ovoid or spherical, some were irregular in form or elongated. The larger bodies were commonly accompanied by small bodies which appeared to originate from the larger by budding. Profiles suggesting binary fission and filamentous forms containing ovoid condensations of cytoplasm were present. The bounding membrane showed the typical trilaminate structure, and DNA-like fibrils were discernible in those MLOs that had an electron lucent central region. In the denser bodies the fibrils were obscured. The MLO ribosomes were distinctly smaller than those in the host cytoplasm. The MLOs were degenerating in phloem cells that were disorganizing and collapsing in response to the infection. Structures in host cells that may be confused with MLO are described.This work was supported by the National Science Foundation grant GB-35228 to K. E and by Hatch and California Statewide Critical Applied Research Funds to the Departmem of Cell Physiology, University of California, Berkeley, California. The authors thank ProfessorJulius H.Freitag for providing the original strains of the aster yellows agent.     

Relation of in vivo morphology to isolation of plant spiroplasmas

Two procedures were developed to isolate plant spiroplasmas directly onto DG-2 agar plates or in DG-2 broth without subcultures or dilutions. The frequency of successful spiroplasma isolations was increased by centrifuging samples, after passing through a 0.45-μm filter, at 25,000 × g for 1 h. Spiroplasmas were obtained from peach, cherry, Madagascar periwinkle, and celery with typical symptoms of the Green Valley strain of X disease (GVX), from peach with typical symptoms of the peach yellow leaf roll strain of X disease (PYLR), from Madagascar periwinkle with typical symptoms of aster yellows (AY), from celery with atypical symptoms of GVX (mild GVX), from plantago with atypical symptoms of aster yellows (mild AY), and from stubborn-diseased citrus. Isolations were consistent (>90%) from plants with mild GVX, mild AY, and citrus stubborn, while isolations were inconsistent (0–9%) from plants with typical symptoms of GVX, PYLR, and AY. The role of the isolated spiroplasmas in plant disease was not determined in this study. All spiroplasma isolates were serologically indistinguishable fromSpiroplasma citri. Spiroplasmas were seen in electron micrographs of 8 out of 9 examined plants from which spiroplasmas were isolated. However, electron micrographs of all 13 examined plants from which no spiroplasmas were isolated contained mycoplasma-like organisms (MLOs) but no, spiroplasmas. These results indicate that there is a correlation between helical MLOs in vivo and successful isolation of spiroplasmas, and that plants may be infected with bothS. citri and nonhelical mycoplasmas.     

MLO-Infected Weeds in the Vineyards of North-western Italy

Ten species of herbaceous plants and shrubs with MLO symptoms such as stunting, bushing, yellowing, reddening, virescence or phyllody were collected in both grapevine yellows (GY)-infected and uninfected vineyards. By electron microscopy, MLOs were found in the sieve tubes of eight species. The presence of MLOs in Picris echioides is demonstrated for the first time. The possible role of MLO-infected weeds in the spread of GY disease is discussed.     

葱兰黄化病病原类菌原体的研究

葱兰为石蒜科(Amaryllidaceae)草本植物,是一种在我国各地普遍栽培的多年生观赏花卉.葱兰黄化病是1992年在杭州发现的一种新病害,该病对葱兰的生长和观赏价值有严重影响.罹病株(丛)最初表现为深红色斑点,逐渐扩大为鲜红色条斑或斑块并转为亮黄色,最后整株叶片大部分转为鲜艳的黄色,呈现典型的系统侵染.发病1—2月后整株(丛)地上部分枯死.发病一般始于早春(3月上旬),春末夏初有一个发病高峰期;秋末有另一个发病期,但病状较轻;感染株有恢复现象,即在下一个生长季能从地下部分重新生长出叶片,但病株叶片明显细小,有丛簇现象,病株开花少且花期短.作者从1992年春起,对该病害进行了病原分离、生物学测定和病组织超薄切片电镜检查,发现该病害可能与类菌原体有关,现将研究结果报告如下.1 材料和方法1.1 样品感病植株分别于1992年4月采自浙江农业大学校园和1992年6月采自杭州花圃,用盆栽保存于防虫温室中,同时采取健株作为对照.1.2 用病株分离病原和接种     

Evolutionary relationships of a plant-pathogenic mycoplasmalike organism and Acholeplasma laidlawii deduced from two ribosomal protein gene sequences.

The families within the class Mollicutes are distinguished by their morphologies, nutritional requirements, and abilities to metabolize certain compounds. Biosystematic classification of the plant-pathogenic mycoplasmalike organisms (MLOs) has been difficult because these organisms have not been cultured in vitro, and hence their nutritional requirements have not been determined nor have physiological characterizations been possible. To investigate the evolutionary relationship of the MLOs to other members of the class Mollicutes, a segment of a ribosomal protein operon was cloned and sequenced from an aster yellows-type MLO which is pathogenic for members of the genus Oenothera and from Acholeplasma laidlawii. The deduced amino acid sequence data from the rpl22 and rps3 genes indicate that the MLOs are more closely related to A. laidlawii than to animal mycoplasmas, confirming previous results from 16S rRNA sequence comparisons. This conclusion is also supported by the finding that the UGA codon is not read as a tryptophan codon in the MLO and A. laidlawii, in contrast to its usage in Mycoplasma capricolum.     

Amplification of plasmid DNA to detect plant pathogenic mycoplasmalike organisms

The 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.     

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.     

Enzymatic activities in cell fractions of mycoplasmalike organisms purified from aster yellows-infected plants.

Mycoplasmalike organisms (MLOs), purified from aster yellows-infected plants were osmotically lysed, and the membranes were separated from the cytoplasmic fraction through differential centrifugation. Electron microscopic examinations of sections of the purified MLOs and the isolated membranes showed pleomorphic bodies and unit membranous empty vesicles, respectively. Cell fractions were tested for NADH oxidase, NADPH oxidase, ATPase, RNase, DNase, and p-nitrophenyl phosphatase activity. NADH oxidase and ATPase were confined to the membrane fraction and NADPH oxidase to the cytoplasmic fraction of the MLOs. para-Nitrophenyl phosphatase, RNase, and DNase activities were detected in both membrane and cytoplasmic fractions, but p-nitrophenyl phosphatase and RNase appeared to be associated with membranes and DNase with the cytoplasmic fraction. Glucose-6-phosphate dehydrogenase was found in the cytoplasmic fraction of the MLO cells. Our findings on the distribution of enzymes in MLO cells and cell fractions are the first basic documentation on nonhelical, nonculturable microbes parasitic to plants.     

Restriction Fragment Length Polymorphisms Within the Apple Proliferation Mycoplasmalike Organism

Genetic 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.     

'Candidatus Lumbricincola', a novel lineage of uncultured Mollicutes from earthworms of family Lumbricidae

Molecular signatures of new, as yet uncultured mollicute-like organisms (MLOs) have been detected in total rRNA and DNA extracted from tissues, gut contents and casts of four species of the earthworm family Lumbricidae. The MLO 16S rRNA sequences exhibited low identity to those of known Mollicutes species and formed a monophyletic cluster distantly affiliated to the ' Candidatus Bacilloplasma' (84.9%) and almost equidistant to the other main phylogenetic group of Mollicutes (< 79.8%) and the classes Bacilli (< 79.5%) and Clostridia (< 76.1%). SSCP profiling and sequence analysis of bands and bacterial clones derived from the earthworms and substrata revealed high phylogenetic relatedness of MLOs in earthworms from different geographic locations (Russia and Germany), with no obvious host species specificity being observed. Fluorescence in situ hybridization (FISH) analysis with a nucleotide probe specific for the new MLO group localized them to the coelomic fluids of earthworms. A new taxonomic group within the Mollicutes , designated ' Candidatus Lumbricincola', is proposed to include these as yet uncultured organisms.     

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.     

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.