Yellows Mycoplasmalike Organism Research Articles

A novel method for cloning DNA of plant-pathogenic mycoplasmalike organisms.

A novel method was developed for cloning the DNA from a representative of plant-pathogenic mycoplasmalike organisms (MLOs). This procedure utilized random amplified polymorphic DNA (RAPD) and basic recombinant DNA techniques. It consisted of amplification of total DNA from diseased plants using one oligonucleotide primer with arbitrary sequence and separation of RAPD products in agarose gels. Unique RAPD band(s) of MLO origin was (were) then recovered from the gel and cloned into the specifically designed vector pCR II. With this method, a DNA fragment of the SA2 isolate of grapevine yellows MLO was cloned. Southern blot hybridizations revealed that most of the DNA in the unique RAPD band was derived from MLO. Results from dot-blot hybridizations used for screening showed that approximately 60% of transformants harbored MLO-specific recombinant plasmids. Our approach is relatively simple, quite efficient, and not limited by the amount of diseased material available. It does not depend on DNA sequence information for primer design and does not rely on restriction endonucleases for cloning. In addition, it can be used directly for disease diagnosis and for differentiation of closely related MLOs. Our system may serve as a model for cloning DNAs of other fastidious plant pathogens.

Detection of the German grapevine yellows (Vergilbungskrankheit) MLO in grapevine, alternative hosts and a vector by a specific PCR procedure

A polymerase chain reaction procedure was developed which enables specific amplification of a ribosomal sequence from the mycoplasmalike organism (MLO) associated with German grapevine yellows (Vergilbungskrankheit, VK) and stolbur-related diseases of solanaceous plants. Successful amplification from all samples prepared from various cultivars collected in different viticultural areas indicates that the causal agent is a relatively homogeneous organism. Amplification was also achieved with template DNA prepared from naturally infected weeds in vineyards such asConvolvolus arvensis andSolanum nigrum, and from the planthopperHyalesthes obsoletus that was collected in the vineyards. Feeding of insects of this species on grapevine seedlings resulted in the development of typical yellows symptoms by the grapes.H. obsoletus could therefore be identified as a vector of Vergilbungskrankheit.

Nature and genetic relatedness of the mycoplasma‐like organism causing rubus stunt in Europe

Cultivated red raspberries (Rubus idaeus), and wild blackberries (R fruticosus, R. caesius, and Rubus hybrids) showing symptoms of rubus stunt were collected in Germany, France, and Italy, Ribosomal DNA of the mycoplasma‐like organism (MLO) that causes rubus stunt was amplified by a polymerase chain reaction procedure and then digested with AtuI and RsaI restriction endonucleases. All samples examined showed the same restriction profiles, which were similar to those of the MLOs inducing elm yellows and alder yellows. However, the rubus stunt MLO could be distinguished from the elm and alder MLOs by Southern blot analysis using DNA probes from a strain of the elm yellows MLO, A variability of the hybridization profiles, probably caused by restriction fragment length polymorphisms, was observed among the rubus stunt samples. From both rDNA restriction site and Southern blot analyses it can be concluded that the rubus stunt agent and the elm yellows and alder yellows MLOs are not identical but closely related and can be grouped in the same taxonomic cluster.

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.

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.

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.

Impact of ash yellows mycoplasmalike organisms on radial growth of naturally infected white, green, and velvet ash

Growth of white ash (Fraxinusamericana L.), green ash (Fraxinuspennsylvanica Marsh.), and velvet ash (Fraxinusvelutina Torr.) as related to infection by ash yellows mycoplasmalike organisms (MLOs) was assessed by diagnosing and grouping trees as MLO infected or healthy and comparing their previous annual radial growth over periods of 9–22 years. White ash and green ash 17–30 years old were studied on two sites per species in central New York State. Velvet ash 30–90 years old was studied in Zion National Park, Utah. MLOs were detected by fluorescence microscopy. Widths of growth rings were measured on increment cores or cross sections of main stems. Growth trends of diseased and healthy groups of white ash and green ash diverged when ash stands were 12–21 years old. In the fifth year after divergence, the unweighted average growth of diseased green ash across two plots was 70% of that of healthy trees. For white ash, the corresponding average was 61%. In white ash tested annually for MLO infection, growth reduction preceded MLO detection by an average of 1 year. Velvet ash did not sustain MLO-associated growth reduction. This species may be tolerant of infection by ash yellows MLOs.

EFFECTS OF ANTIBIOTIC INJECTION ON ASH YELLOWS-INFECTED WHITE ASH (FRAXINUS AMERICANAL.)

Summary Fifteen white ash (Fraxinus americana L.) naturally infected with ash yellows (AY) Mycoplasma-like organism (MLO) were injected twice, in September 1986 and June 1987, with 4 per cent Oxytetracycline (OTC) using Mauget trunk injection (average 1.2 g active ingredient (a.i.)/tree). MLOs were not detected in the twigs or in the roots for 30 days following OTC injection, then increased gradually to the control level. Fifteen more AY-infected white ashes were injected in June 1987 with 4 per cent OTC Mauget injection at variable dosages (averages 1.2, 1.5, 2.4 g a.i./tree). Higher dosages of OTC decreased the MLO population for approximately two months. Similar injections of penicillin and of streptomycin had no effect on MLO populations. OTC therapy may be the most efficient at high rates on trees without decline symptoms, i.e. early in disease progression.

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)

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 [...]

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 [...]

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 [...]

Production of Monospecific Polyclonal Antibodies Against Aster Yellows Mycoplasmalike Organism-Associated Antigen

Proteins of mycoplasmalike organisms (MLO) associated with aster yellows were detected by Western blotting in partially purified preparations from aster yellows-infected plants. Polyclonal antiserum produced against aster yellows (AY) MLO recognized a 23-kDa protein in AY-infected plants that was not found in healthy controls. In addition, this antiserum recognized proteins common to AY-infected and healthy samples. Cross-absorption of the PA with healthy plant extracts did not completely eliminate the nonspecific antibodies. Antibodies specific for AY MLO-associated protein were purified from polyclonal antibodies by trapping them on AY MLO protein obtained by electrophoresis of infected plant extracts and transferred onto nitrocellulose [...]

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

Comparison of Monoclonal Antibodies, DNA Probes, and PCR for Detection of the Grapevine Yellows Disease Agent

Two monoclonal antibodies (MAbs) specific for a grapevine yellows mycoplasmalike organism (GY-MLO) were produced by fusing mouse myeloma cell lines NS1/1 to spleen cells of mice immunized with partially purified GY-MLO from diseased periwinkles. Using enzyme-linked immunosorbent assays (ELISAs) and indirect immunofluorescence (IF) staining, the MAbs were shown to be specific for the GY-MLO. Although both ELISA and IF tests were satisfactory with diseased periwinkles, serological detection of GY-MLO in diseased grapevines was not always reliable because of the extremely low titer of GY-MLO in the phloem and the interference of plant pigments in dot blot immunoassays [...]

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

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.

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.