Aster Yellows Mycoplasmalike Organism Research Articles

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.

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.

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.

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.

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

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

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

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

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

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

Detection of a Plant Pathogen in a Nonvector Insect Species by the Polymerase Chain Reaction

A nonculturable mycoplasmalike plant pathogen was detected in a leafhopper species capable of transmitting the pathogen as well as in a leafhopper species that does not vector the pathogen. Amplification of a pathogen-specific DNA sequence by polymerase chain reaction (PCR) revealed the presence of pathogen DNA in total nucleic acid extracts of the vector insect Macrosteles fascifrons and of the nonvector insect Dalbulus maidis, both of which had fed on plants infected by the aster yellows mycoplasmalike organism

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

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

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

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

Sensitive detection of mycoplasmalike organisms in field‐collected and in vitro propagated plants of Brassica, Hydrangea and Chrysanthemum by polymerase chain reaction

SummaryA polymerase chain reaction (PCR) protocol, previously designed for amplification of a DNA fragment from aster yellows mycoplasmalike organism (MLO), was employed to investigate the detection of MLO DNA in field‐collected and in vitro micropropagated plants. PCR with template DNA extracted from symptomatic, naturally‐infected samples of Brassica, Chrysanthemum and Hydrangea, each yielded a DNA band corresponding to 1.0 Kbp. However, no DNA product was observed when either infected Ranunculus (with phyllody disease) or Gladiolus with (symptoms of ‘germs fins’) was used as source of template nucleic acid for PCR; further experiments indicated absence of target DNA in the case of Ranunculus and the presence of substances in Gladiolus which inhibited the PCR. The MLO‐specific DNA was detected by PCR using less than 95 pg of total nucleic acid (equivalent to total nucleic acid from 1.9, ug tissue) in the case of field‐collected Hydrangea and less than 11.4 pg of nucleic acid (equivalent to total nucleic acid from 19 ng of tissue) in the case of field‐collected Brassica. The findings illustrate highly sensitive detection of MLOs in both field‐grown and in vitro micropropagated infected plants.

Sensitive detection and identification of mycoplasma-like organisms in plants by polymerase chain reactions

DNA amplification by polymerase chain reactions (PCR) was employed to detect host plant infection by several mycoplasmalike organisms (MLOs), including the aster yellows (AY), dwarf aster yellows (DAY), and periwinkle little leaf (0-1) MLOs. For PCR, two pairs of oligonucleotide primers, designated AY18pm and AY19pm, respectively, were synthesized on the basis of partial sequences of cloned AY MLO DNA fragments AY18 and AY19. Reaction mixtures containing primer pair AY18pm yielded a DNA product of 1.6Kbp, when template consisted of DNA extracted from AY MLO- or DAY MLO-infected Catharanthus roseus (periwinkle). A DNA product of 1.0Kbp was obtained with primer pair AY19pm, when template consisted of DNA extracted from C. roseus infected by AY MLO, DAY MLO, or periwinkle little leaf (strain O-1) MLO. MLO-specific bands were observed when reaction mixtures contained as little as 5 pg total nucleic acid from infected plants. No PCR product was observed when reaction mixtures contained only DNA from healthy plants or DNA from plants infected by western X MLO or by tomato big bud MLO. The findings indicated that the PCR system is useful for sensitive detection and differentiation of MLOs in infected hosts.

Phylogenetic relationships between the western aster yellows mycoplasmalike organism and other prokaryotes established by 16S rRNA gene sequence.

Restriction fragments containing the 16S rRNA gene of the western aster yellow mycoplasmalike organism (SAY-MLO) were identified in Southern blots probed with cloned fragments of the western X-disease mycoplasmalike organism 16S rRNA gene. Two fragments which contained the entire SAY-MLO 16S rRNA gene and flanking DNA were cloned in M13 and sequenced. The SAY-MLO 16S rRNA gene is approximately 1,535 bp long, has a G+C content of 47 mol%, and has an overall secondary structure similar to that proposed for Escherichia coli. Putative rRNA promoter sequences and sequences involved in processing of the primary rRNA transcript were similar in the SAY-MLO, two Mycoplasma species, and Bacillus subtilis, suggesting that these prokaryotes and the mycoplasmalike organisms may have similar transcriptional and processing enzymes. We identified two tRNA genes, a tRNA(Tyr) (GTA) gene upstream from the 16S rRNA gene and a tRNA(Ile) (GAT) gene in the spacer region between the 16S and 23S rRNA genes. Comparisons of the SAY-MLO 16S rRNA nucleotide sequence with 16S rRNA sequences of other organisms indicated that the SAY-MLO is phylogenetically related most closely to other plant-pathogenic mycoplasmalike organisms, followed by Anaeroplasma species, Acholeplasma species, and some Mycoplasma species.

A Genotype-Based System for Identification and Classification of Mycoplasmalike Organisms (MLOs) in the Aster Yellows MLO Strain Cluster

Fifteen mycoplasmalike organism (MLO) strains from North America and Europe, including aster yellows (AY), tomato big bud (BB), clover phyllody (CPh), chrysanthemum yellows (CY), and unknown MLOs, were studied. These MLOs are among those previously identified, on the basis of dot hybridizations, as members of the AY MLO strain cluster. Restriction fragment length polymorphism (RFLP) analyses revealed that all 15 strains can be classified into three genomic types: type I (typified by eastern AY MLOs), type II (e.g., western AY and CY MLOs), and type III (e.g., CPh MLO) (...)

Distribution and Multiplication of Western Aster Yellows Mycoplasmalike Organisms inCatharanthus roseusas Determined by DNA Hybridization Analysis

Mycoplasmalike organism (MLO) specific DNA probes derived from chromosomal or plasmid DNA of the severe strain of western aster yellows MLO (SAY) were used to monitor the distribution and multiplication of MLOs in periwinkle plants infected with the SAY or dwarf strain (DAY) of the western AY MLO. Plants were graft-inoculated, and DNA was extracted from different regions of the inoculated plants over a 10-wk period. DNA samples were applied to nitrocellulose membranes and hybridized to cloned, 32 P-labeled, MLO-specific DNA probes (...)

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.

Localization of Pathogenic Mycoplasma-like Organisms in Plant Tissue Using in situ Hybridization.

A cloned chromosomal DNA fragment of clover proliferation mycoplasmalike organism (MLO) was sequenced and used to synthesize biotinylated single-stranded DNA probes by asymmetric polymerase chain reaction. The biotinylated single-stranded DNA was identified by temperature gradient gel electrophoresis. Hand sections were prepared from tissues of periwinkle plants (Catharanthus roseus (L.) G. Don) individually inoculated with clover proliferation MLO or potato witches'-broom MLO. In situ hybridization of the sections with the biotinylated probes showed that clover proliferation MLO and potato witches'-broom MLO were located mainly in sieve tube elements. Western aster yellows MLO was not detected by in situ hybridization with the same biotinylated probes. MLO was detected first in the external phloem tissue and then in the internal phloem tissue as plants grew older. This investigation provides the first example of developing PCR-generated small biotinylated single-stranded DNA to trace the colonization of MLOs in plant tissues.

DNA Hybridization Between Western Aster Yellows Mycoplasmalike Organism Plasmids and Extrachromosomal DNA from Other Plant Pathogenic Mycoplasmalike Organisms

DNA Hybridization Between Western Aster Yellows Mycoplasmalike Organism Plasmids and Extrachromosomal DNA from Other Plant Pathogenic Mycoplasmalike Organisms

Differentiation of virescence MLOs using western aster yellows mycoplasma-like organism chromosomal DNA probes and restriction fragment length polymorphism analysis

Summary: Two chromosomal fragments of the severe strain (SAY) of the western aster yellows mycoplasma-like organism (MLO) were cloned in Escherichia coli. These fragments were used to probe Southern blots of DNA extracted from plants infected with geographically and pathologically diverse MLOs. The two SAY probes hybridized with some, but not all, of the virescence-inducing MLOs examined, indicating that some of these MLOs are genetically related. Comparisons of restriction fragment length polymorphisms of MLO DNA established additional relationships within this group. The SAY-MLO probes did not hybridize with decline-inducing MLOs, plant pathogenic spiroplasmas, or healthy plants.

Identification and characterization of plasmids from the western aster yellows mycoplasmalike organism

Supercoiled double-stranded DNA molecules (plasmids) were isolated from plants infected with three laboratory strains of western aster yellows mycoplasma-like organism (AY-MLO) by using cesium chloride-ethidium bromide density gradients. Southern blot analysis, using plasmids from the severe strain of AY-MLO (SAY-MLO) as the probe, identified at least four plasmids in celery, aster, and periwinkle plants and in Macrosteles severini leafhopper vectors infected with either the dwarf AY-MLO, Tulelake AY-MLO, or SAY-MLO strain. Plasmids were also detected in two California field isolates of AY-MLO but not in plants infected with the beet leafhopper-transmitted virescence agent, western X, or elm yellows MLOs. SAY-MLO plasmids were 5.2, 4.9, 3.4, and 1.7 kilobase pairs in size. Plasmids isolated from dwarf AY- and Tulelake AY-MLOs were 7.4, 5.1, 3.5, and 1.7 kilobase pairs in size. No evidence was obtained for integration of SAY-MLO plasmids into the MLO chromosome.