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.     

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.     

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.     

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.     

Susceptibility of Grafted Malus Taxa and Hybrids to Apple Proliferation Disease

Seedlings of a great number of Malus species, subspecies, cultivars and hybrids were graft-inoculated with the apple proliferation MLO. The scion cultivars were M. pumila cv.‘Cox's Orange Pippin’and‘Golden Delicious’. The grafted trees responded very differently to infection. According to recovery rate, witches’broom formation, mortality, and development of the MLO population, the tested material could be divided into 5 groups. Group I corresponds to the domestic apple M. pumila and is characterized by a low recovery rate, a low mortality, a high frequency of witches’broom formation, and a high MLO titer. Group II differs from group I mainly by a higher mortality. In group III, mortality is like in group II but recovery is higher while witches’broom formation and MLO titer are significantly lower. Group IV is characterized by a mortality of more than 50 %. Both witches’broom formation and the numbers of MLOs in the phloem are usually low. In group V, most of the trees recovered or showed never symptoms while mortality was low. After inoculation the MLO population was low or appeared degenerate. During recovery the number of MLO-positive samples decrease so that by the end of the observation period the organisms could not longer be detected in most cases. Group V consists of apomictic rootstock selections deriving from crosses of M. sieboldii and M. sargentii with M. pumila. Due to the combination of low mortality with the apparent elimination of the MLOs within a few years this group fulfills the requirement of resistant rootstocks suitable for controlling apple proliferation.     

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.     

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.     

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.     

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.     

Culture-independent analysis of bacterial species from an anaerobic mat from Lake Fryxell, Antarctica: prokaryotic diversity revisited.

Using a lake sediment mat sample from Lake Fryxell, Antarctica, different DNA extraction and purification methods were compared by denaturing gradient gel electrophoresis (DGGE). Based on the analyses of cloned 16S rRNA gene sequences a high degree of as yet uncultured prokaryotes have been reported in this sample. Although the vast majority of these as yet uncultured organisms seem to be classified as representatives of Firmicutes, Proteobacteria and Bacteriodetes, many of these taxa should be regarded novel species as judged from the distance of their gene sequences to those of their nearest cultured phylogenetic neighbours. The physiological properties of cultured strains from Lake Fryxell and of those of described species that are phylogenetically affiliated to the as yet uncultured species from this environment, suggest the presence of a well developed food web of primary producers, anaerobic degraders and fermenters, and aerobes. The few novel species described from this sample add to the increasing number of species characterized from various Antarctic habitats. Determination of the phylogenetic relatedness of the mat clone sequences of Clostridia with recent entries into public databases revealed that many of the putative species are closely related to other putative species detected in a broad range of environments, ranging from rumen and gut, anaerobic and polluted soil to sediment and groundwater samples.     

16S rRNA gene-based oligonucleotide microarray for environmental monitoring of the betaproteobacterial order "Rhodocyclales"

For simultaneous identification of members of the betaproteobacterial order "Rhodocyclales" in environmental samples, a 16S rRNA gene-targeted oligonucleotide microarray (RHC-PhyloChip) consisting of 79 probes was developed. Probe design was based on phylogenetic analysis of available 16S rRNA sequences from all cultured and as yet uncultured members of the "Rhodocyclales." The multiple nested probe set was evaluated for microarray hybridization with 16S rRNA gene PCR amplicons from 29 reference organisms. Subsequently, the RHC-PhyloChip was successfully used for cultivation-independent "Rhodocyclales" diversity analysis in activated sludge from an industrial wastewater treatment plant. The implementation of a newly designed "Rhodocyclales"-selective PCR amplification system prior to microarray hybridization greatly enhanced the sensitivity of the RHC-PhyloChip and thus enabled the detection of "Rhodocyclales" populations with relative abundances of less than 1% of all bacteria (as determined by fluorescence in situ hybridization) in the activated sludge. The presence of as yet uncultured Zoogloea-, Ferribacterium/Dechloromonas-, and Sterolibacterium-related bacteria in the industrial activated sludge, as indicated by the RHC-PhyloChip analysis, was confirmed by retrieval of their 16S rRNA gene sequences and subsequent phylogenetic analysis, demonstrating the suitability of the RHC-PhyloChip as a novel monitoring tool for environmental microbiology.     

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.     

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.     

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.     

Histopathology of Apple Proliferation in Malus Taxa and Hybrids of Different Susceptibility

Malus taxa and hybrids (“taxa”) grafted with M. pumila cv.‘Golden Delicious’differ significantly in their susceptibility to apple proliferation which is caused by a mycoplasma-like organism (MLO). These differences are correlated with the severity of anatomical aberrations and the numbers of MLOs in the phloem. The roots of declining trees of highly susceptible taxa with a mortality of more than 50 % are characterized by extensive phloem necrosis and the depletion of starch. MLOs are either not detectable or are present in low numbers, or the population appears degenerate when viewed by fluorescence microscopy. In comparable trees of a hybrid of M. sieboldii×M. pumila which shows a high recovery rate, both phloem necrosis and starch depletion are less pronounced, and the MLO numbers are low or the organisms are not detectable. Decline-tolerant taxa such as M. silvestris or M. pumila×M. baccata are little affected. Their phloem conditions and starch content do not differ significantly from that of healthy trees. However, the MLO titer is high. The histopathology of the scion cultivar of all groups examined is rather similar to that of the roots of the decline-tolerant taxa. Only in a late stage of decline, phloem necrosis increases while starch content and MLO numbers decrease in the scions grafted onto highly susceptible stockls.     

16S rRNA Gene-Based Oligonucleotide Microarray for Environmental Monitoring of the Betaproteobacterial Order “Rhodocyclales”

For simultaneous identification of members of the betaproteobacterial order “Rhodocyclales” in environmental samples, a 16S rRNA gene-targeted oligonucleotide microarray (RHC-PhyloChip) consisting of 79 probes was developed. Probe design was based on phylogenetic analysis of available 16S rRNA sequences from all cultured and as yet uncultured members of the “Rhodocyclales.” The multiple nested probe set was evaluated for microarray hybridization with 16S rRNA gene PCR amplicons from 29 reference organisms. Subsequently, the RHC-PhyloChip was successfully used for cultivation-independent “Rhodocyclales” diversity analysis in activated sludge from an industrial wastewater treatment plant. The implementation of a newly designed “Rhodocyclales”-selective PCR amplification system prior to microarray hybridization greatly enhanced the sensitivity of the RHC-PhyloChip and thus enabled the detection of “Rhodocyclales” populations with relative abundances of less than 1% of all bacteria (as determined by fluorescence in situ hybridization) in the activated sludge. The presence of as yet uncultured Zoogloea-, Ferribacterium/Dechloromonas-, and Sterolibacterium-related bacteria in the industrial activated sludge, as indicated by the RHC-PhyloChip analysis, was confirmed by retrieval of their 16S rRNA gene sequences and subsequent phylogenetic analysis, demonstrating the suitability of the RHC-PhyloChip as a novel monitoring tool for environmental microbiology.     

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.     

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.     

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.     

Base Composition of the DNA of Mycoplasma-Like Organisms Associated with Various Plant Diseases

DNA was isolated from periwinkle plants infected by various mycoplasma-like organisms (MLOs), and from apple trees affected by apple proliferation. The DNA of the causal agents was separated from the host plant DNA by repeated bisbenzimide-CsCl buoyant density gradient centrifugation which resulted in highly enriched MLO DNA bands characterized by a lower buoyant density than that of the host DNA. The MLO DNAs were hydrolyzed to free bases which were determined by HPLC. The analyses revealed a similar low G + C content as found in the DNAs of several culturable mycoplasmas and spiroplasmas. The values of the DNA of the agents of the diseases investigated were as follows: European aster yellows 23.0, periwinkle virescence 23.5, apple proliferation 23.7, rape virescence 24.2, and phyllody of Diplotaxis erucoidcs 26.2 mol % G + C, respectively. Methylated bases were detected in low amounts only.