Yellows Mycoplasmalike Organism Research Articles

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 (...)

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) (...)

Cloned DNA Probes Specific for Detection of a Mycoplasmalike Organism Associated with Ash Yellows

DNA was isolated from periwinkle (Catharanthus roseus) infected with a mycoplasmalike organism (MLO) that originated in white ash (Fraxinus americana) affected by ash yellows. The DNA was digested with EcoRI and HindIII, ligated to plasmid vector pIBI30, and cloned in Escherichia coli DH5.alpha.. Cloned DNA inserts were excised from four ash yellows-specific recombinant plasmids, labeled with biotin, and employed as probes in dot and Southern hybridizations. None of the probes hybridized with nucleic acid from healthy plants; all hybridized with nucleic acid from periwinkle infected by the ash yellows MLO. Southern hybridization analyses showed the probes to contain MLO chromosomal DNA. In dot hybridizations performed at 42.degree.C, probe AA131 hybridized only with nucleic acid from plants infected by the ash yellows MLO, whereas the other three probes, designated AA82I, AA157I, and AA176I, hybridized with nucleic acid from plants infected by any of several MLOs, including the ash yellows MLO. Under conditions of high stringency (52.degree.C), all four probes hybridized only with nucleic acid from plants infected by the ash yellows MLO. In diagnostic tests on naturally diseased plants, probes AA13I and AA176I hybridized with nucleic acid extracted from leaves, twigs, trunk phloem, and roots of white ash with symptoms of ash yellows but not with nucleic acid from healthy ash grown from seed. The findings provide means for specific detection and identification of ash yellows MLO and support the concept that this MLO represents a distinct strain cluster.

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.

SYMPTOM DEVELOPMENT OF ASH YELLOWS AND FLUCTUATION OF MYCOPLASMA-LIKE ORGANISM POPULATION IN WHITE ASH (Fraxinus americana L.)

Summary White ashes (Fraxinus americana) naturally infected with ash yellows mycoplasma-like organism (MLO) were observed monthly, from September 1986 to August 1989. The symptoms were witches'-broom (WB), foliar chlorosis (CHL), deliquescent branching (DB), tufting (TF), premature spring bud break (PSB), and premature autumn colour (PAC). Every August each tree was rated for decline in less vs. more than half the crown. All symptoms except PSB and PAC differed between these two groups. In more than the 1 and 2 groups, incidence of WB, CHL and TF increased significantly each year, but DB, PSB and PAC did not. WB and TF were the strongest indicator of ash yellows even though those symptoms were found at the late stage of the disease. MLO was verified monthly by DAPI staining. MLO population fluctuated in the infected tree over time. In twigs, MLO population was high during the growing season and quite low during the dormant season. In contrast, the MLO population in root tissues was highest during the dorm...

Ultrastructural features of primula yellows mycoplasma-like organism (MLO) in cryosections of Catharanthus roseus leaves

Cryoultramicrotomy was applied to the study of primula yellows (PY) mycoplasma-like organism (MLO) in Catharanthus roseus leaves. The aim was to check whether this method could give better information on morphology and structure of MLOs, in comparison to conventional Epon-Araldite embedding. After aldehyde fixation, some of the samples were processed for conventional sectioning, and others were frozen in liquid propane and cryosectioned. Two predominant morphotypes were observed, spherical and filamentous, often associated or connected to each other. MLO structural details were better preserved in cryosections. The electron transparency of cryosections even at a thickness of 200 nm made it easy to follow the morphology and spatial development of the filaments. The filaments were tubular, 35–45 nm in diameter and about 1.2 μm long, or were partially or completely pinched into long chains of discrete oval or spherical bodies, 45–75 nm in diameter. The rounded MLOs, 200–250 nm in diameter, were sometimes connected in a chain by a continuous outer membrane. These observations suggest that the filamentous forms may be involved in growth and reproduction of the PY MLO, in a manner similar to that shown for analogous structures observed in other plant MLOs and animal mycoplasmas.

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.

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.

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.

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.

PURPLE TOP AS A CAUSE OF POTATO CHIP DISCOLORATION

ABSTRACT Purple top (PT) a disease in potatoes. Solarium tuberosum L. caused by the aster yellows mycoplasma-like organism (AY-MLO) was shown to cause chip browning in processed potatoes. PT was produced in field experiments from 1984-1987, in which infective aster leafhoppers were caged on plants of the potato cvs. Monona and Norchip. Tubers from these plots, tubers from plants exposed to non-infective leafhoppers, and tubers from non-caged controls were harvested, stored, and samples were chipped at monthly intervals. Chips from PT-infected tubers appeared normal until about the January-February processing when dark discoloration occurred in the area of the vascular ring. In March through June chippings, the brown discoloration had spread and intensified in chips in the medullary and cortical areas of chips. Chips from tubers of plants exposed to healthy aster leafhoppers and from tubers from non-caged plants, on average, retained a bright color. The browning was similar in chips obtained from PT-infected tubers stored at 9° or 11° C and may be due to excessive accumulation of reducing sugars resulting in non-enzymatic browning.

Detection of Chrysanthemum Yellows Mycoplasmalike Organism by Dot Hybridization and Southern Blot Analysis

(...) Of five probes used in Southern blot analyses, one probe containing extrachromosomal DNA from O-1 MLO hybridized with extrachromosomal but not chromosomal DNA of CY MLO. Three probes containing chromosomal DNA of AY MLO and one containing chromosomal DNA of O-1 MLO hybridized with chromosomal DNA of CY MLO. The nucleotide sequence homologies between the probes and nucleic acid of CY MLO are evidence for genetic relatedness of a European MLO and American MLOs and provide diagnostic tools for CY MLO detection

Preparation of mycoplasma immunogens from plants and a comparison of polyclonal and monoclonal antibodies made against primula yellows MLO‐associated antigens

SummaryA method is described for obtaining from plants partially purified preparations of mycoplasma‐like organisms (MLO) which are suitable for use as immunogens for polyclonal or monoclonal antibody production, and as antigens for directly coating ELISA plates. Using this method a mouse monoclonal antibody to primula yellows MLO was prepared, and its characteristics compared with those of primula yellows polyclonal antibodies from rabbits and also against polyclonal antibodies made to similar preparations of European aster yellows MLO. No serological distinction was obtained between any of the homologous or heterologous combinations of antibody and MLO preparation using ELISA, fluorescence microscopy with FITC‐labelled antibodies, or immunoprobes of western blots of partially purified MLO preparations. By contrast, there were no cross‐reactions between the primula or aster yellows antibodies or MLO preparations and preparations of clover phyllody or tomato big bud MLOs or their respective polyclonal antibodies. The primula yellows MLO monoclonal and polyclonal antibodies, and also the European aster yellows MLO polyclonal antibodies, all appeared to recognize only a single major antigen of approximate M, = 22 400 daltons. Some possible explanations for the apparent specificity of the polyclinic antisera for a single antigen, and the relevance to MLO preparation procedures are discussed.

アネモネてんぐ巣病（新称）の発生とその宿主範囲

In 1986, a disease of anemone plant, showing the symptoms of stunting and witches' broom, was observed in Fukude, Shizuoka prefecture. Electron microscopy revealed the presence of mycoplasmalike organisms (MLOs) in the sieve tubes of the diseased plant. The MLO were transmitted to healthy anemone and garland chrysanthemum by the leafhopper Macrosteles striifrons Anufriev. Of 34 species plants in 15 families, which were inoculated by the infectious M. striifrons, 31 species plants in 13 families were infected with the MLO. Anemone witches' broom MLO described in this work had a wide host range similar to that of onion yellows MLO.

ヒメフタテンヨコバイが媒介するトマト萎黄病

A disease of tomato, showing the symptoms of yellows and stunting, was found in Saga and Hiroshima Prefectures in 1983 and 1984, respectively. Electron microscopy revealed the presence of numerous mycoplasmalike organisms (MLOs) in the phloem tissues of the diseased plants. Of the two leafhoppers tested, Macrosteles orientalis Virbaste and Scleroracus fiavopictus Ishihara, only M. orientalis transmitted the disease. Tomato yellows MLO described in this work had a wide host range similar to that of onion yellows MLO.

Comparison of Monoclonal Antibodies and Polyclonal Antibodies in Detection of the Aster Yellows Mycoplasmalike Organism

Comparison of Monoclonal Antibodies and Polyclonal Antibodies in Detection of the Aster Yellows Mycoplasmalike Organism

Aster Leafhopper and Aster Yellows Control in Carrot, 1984

Abstract Three insecticides were evaluated to determine their effects on the aster leafhopper and its transmission to aster yellows (mycoplasma-like organism) to carrots. The trial was conducted at the University of Minnesota Agricultural Experiment Station, Rosemount, MN. Each plot had eight 20-ft rows (12-inch centers), and treatments were arranged in a randomized complete block with 4 replicates. Both sides of each plot were bordered by 2 rows of lettuce, Lactuca sativa L., ‘Great Lakes’ which remained unsprayed and served as a source of aster leafhopper and aster yellows. Individual plots were separated from others by 10 ft of bare ground on all sides to minimize interplot movement of the aster leafhopper. Treatments were applied using a CO2 powered backpack sprayer at 40 psi (57.5 gal/acre). Application dates were 18, 24, 31 Jul and 16 Aug. Sweep samples were taken with a 15-inch diam sweep net taking 25 sweeps per plot.

Aster Leafhopper and Aster Yellows Control in Carrot, 1983

Abstract Three insecticides were evaluated to determine their effects on the aster leafhopper and its transmission of aster yellows (mycoplasma-like organism) to carrots. The trial was conducted at the Univ. of Minnesota Agric. Exp. Stn., Rosemount, Minn. Each plot had two 20-ft rows (40-in. centers) of ‘Scarlet Nantes’ (aster yellows resistant) and ‘Nantes Coreless’ (aster yellows susceptible) and treatments were arranged in a randomized complete block with four replicates. Both sides of each plot were bordered by two rows of lettuce, Lactuca sativa L., ‘Great Lakes’ which remained unsprayed and served as a source of aster leafhopper and aster yellows. Individual plots were separated from others by 10 ft of bare ground on all sides to minimize interplot movement of ickpack sprayer at 30 lb/in2 (27.0 the aster leafhopper. Treatments were applied using a CO2 powered backpack sprayer at 30 lb/in2 (27.0 gal/acre) except for Sevin XLR which was applied at 15 lb/inz (11.5 gal/acre). Treatments were applied biweekly from 30 June to 4 Aug. and weekly from 4 Aug. through 25 Aug. Fourteen applications in all were made. Sweep samples were taken with a 15-in. diameter sweep net (25 sweeps per plot). Insect counts were taken from all four rows, but not separated with respect to cultivar.

Maintenance In Vitro of the Aster Yellows Mycoplasmalike Organism

Maintenance In Vitro of the Aster Yellows Mycoplasmalike Organism