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 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.     

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.     

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.     

Identification and grouping of mycoplasmalike organisms associated with grapevine yellows and clover phyllody diseases based on immunological and molecular analyses.

Immunofluorescent staining, dot blot hybridization, PCR, random amplified polymorphic DNA (RAPD) markers, and restriction fragment length polymorphism wee used to study the genetic relatedness among mycoplasmalike organisms (MLOs) associated with several geographically diverse grapevine yellows diseases (CA1, CH1, SA1, and SA2 from Bologna, Italy; GYU from Udine, Italy; GYR from Rome, Italy; and GYG from Germany). The relationship between these and MLOs associated with clover phyllody diseases in Italy (CPhB and CPhC) and Canada (CPhCa) was also examined. Two monoclonal antibodies reacted with MLOs of GYU-, CPhB-, and CPhC-infected periwinkles. Dot blot hybridization with two cloned GYU DNA fragments, GYD-1 and GYD-2 inserts, showed that both hybridized with DNAs of GYU-, CPhB-, and CPhC-infected periwinkles but not with those of GYR and CPhCa. In addition, GYD-1 insert hybridized with DNAs of CA1, CH1, SA1, SA2, and GYG. Three primer pairs were developed in PCR experiments for this study. By using primer set GYD2P1F and GYD2P1R, a 600-bp DNA fragment was amplified only when DNAs from GYU-, CPhB-, and CPhC-infected plants were used as templates. With the primer pair GYD2P1F and GYD2P2R, a 550-bp DNA fragment was amplified from GYU, CPhB, CPhC, and GYG. The primer pair GYD1P1F and GYD1P2R, on the other hand, could amplify all isolates, although the patterns of PCR products were not identical for all isolates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Two Intracellular Symbiotic Bacteria from the Mulberry Psyllid Anomoneura mori (Insecta, Homoptera)

We characterized the intracellular symbiotic bacteria of the mulberry psyllid Anomoneura mori by performing a molecular phylogenetic analysis combined with in situ hybridization. In its abdomen, the psyllid has a large, yellow, bilobed mycetome (or bacteriome) which consists of many round uninucleated mycetocytes (or bacteriocytes) enclosing syncytial tissue. The mycetocytes and syncytium harbor specific intracellular bacteria, the X-symbionts and Y-symbionts, respectively. Almost the entire length of the bacterial 16S ribosomal DNA (rDNA) was amplified and cloned from the whole DNA of A. mori, and two clones, the A-type and B-type clones, were identified by restriction fragment length polymorphism analysis. In situ hybridization with specific oligonucleotide probes demonstrated that the A-type and B-type 16S rDNAs were derived from the X-symbionts and Y-symbionts, respectively. Molecular phylogenetic analyses of the 16S rDNA sequences showed that these symbionts belong to distinct lineages in the γ subdivision of the Proteobacteria. No 16S rDNA sequences in the databases were closely related to the 16S rDNA sequences of the X- and Y-symbionts. However, the sequences that were relatively closely related to them were the sequences of endosymbionts of other insects. The nucleotide compositions of the 16S rDNAs of the X- and Y-symbionts were highly AT biased, and the sequence of the X-symbiont was the most AT-rich bacterial 16S rDNA sequence reported so far.     

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.     

Detection and Characterization of Phytoplasma and Sugarcane Yellow Leaf Virus Associated with Leaf Yellowing of Sugarcane

Leaves from sugarcane were collected from Egyptian plantation fields and tested for phytoplasma (Sugarcane yellows phytoplasma, SCYP) and Sugarcane yellow leaf virus (SCYLV) using nested PCR (with different primers) and RT‐PCR, respectively. These results showed significant differences in the amplification of the PCR assays. The primer MLO‐X/MLO‐Y, which amplified the 16S‐23S rDNA spacer region, was the most precise to detect the phytoplasma in sugarcane plants. Sequencing and restriction fragment length polymorphism analysis revealed that all tested phytoplasmas belonged to the 16SrI (aster yellows phytoplasma) group, with the exception of cultivar G84‐47 belonged to the 16SrXI (Rice yellow dwarf phytoplasma) group. Three Egyptian sugarcane cultivars were phytoplasma free. Phylogenetic analyses of 34 screened accessions of 16S ribosomal DNA gene sequences of Candidatus phytoplasma including the ones collected from Egypt used in this study and those extracted from GenBank showed that they split into two distinct clusters. The phylogenetic analyses indicated that these phytoplasmas are closely related and share a common ancestor. All tested Egyptian sugarcane plants were infected by SCYLV with the exception of cultivar Phil‐8013 which was virus free.     

Detection and Identification of a 16SrII Group Phytoplasma Causing Clover Little Leaf Disease in Iran

White clover plants showing little leaf and leaf reddening symptoms were observed in Isfahan Province in central Iran. Restriction fragment length polymorphism analyses of nested PCR‐amplified fragments from Iranian clover little leaf phytoplasma isolates and representative phytoplasmas from other phytoplasma groups using AluI, CfoI, KpnI and RsaI restriction enzymes indicated that the clover phytoplasma isolates are related to the peanut WB group. Sequence analyses of partial 16S rRNA fragments showed that Iranian clover little leaf phytoplasma has 99% similarity with soybean witches'‐broom phytoplasma, a member of the peanut WB (16SrII) phytoplasma group. This is the first report of clover infection with a phytoplasma related to the 16SrII group.     

Isolation, characterization, and physical localization of 33 human X-chromosome RFLP markers

In a search for highly polymorphic X-specific loci, the X-chromosome DOE Ch35 phage library (LAOXNL01) was screened with three oligonucleotides representative of minisatellite consensus sequences. A total of 170 clones containing human inserts were isolated by hybridization to the oligonucleotide sequences; each was tested for polymorphism on five random female DNAs with six restriction enzymes. Among the 53 clones demonstrating a polymorphic pattern, 47 were of distinct origin. Twelve of the polymorphisms (23%) were determined to be autosomal. Polymorphisms for the remaining 35 clones were characterized, These polymorphisms represent 33 new X-chromosome RFLP loci, since two pairs of clones detected partially overlapping patterns. A pattern of similar length variation with multiple enzymes ("VNTR-type") was demonstrated in 6 (50%) of the 12 non-X-polymorphic clones. However, only 3 (9%) of the 33 X polymorphic loci showed VNTR-like patterns, suggesting a decreased amount of VNTR polymorphism on the X chromosome. The 33 polymorphic X loci were physically localized with a set of rodent x human somatic cell hybrid DNAs representing nine different X-chromosome breakpoints.     

New informative polymorphism at the DXS304 locus,a close distal marker for the fragile X locus

Summary The polymorphic DNA marker DXS304 detected by probe U6.2 has recently been shown to be closer to the fragile X locus than previously available markers. Its usefulness has however been limited by its relatively low heterozygosity. We have isolated, by cosmid cloning, a 67 kilobase region around probe U6.2 and have characterized a new probe (U6.2-20E) that detects BanI and BstEII restriction fragment length polymorphisms (RFLPs). The BanI RFLP has a heterozygosity of 0.49 and is in partial linkage disequilibrium with the previously described polymorphism, with a combined heterozygosity of 0.63. Furthermore, we have found that the U6.2 original probe, which probably detects an insertion-deletion polymorphism, is also informative in BanI digests. Thus, the two informative RFLPs at the DXS304 locus can be conveniently tested in a single hybridization with a single digest. An updated linkage analysis confirms that DXS304 is distal to the fragile X locus. This informative locus can now be used effectively for genetic mapping of the Xq27–q28 region, and for diagnostic applications in fragile X or Hunter syndrome families.     

Differentiation of mycoplasmalike organisms (MLOs) in European fruit trees by PCR using specific primers derived from the sequence of a chromosomal fragment of the apple proliferation MLO.

A 1.8-kb chromosomal DNA fragment of the mycoplasmalike organism (MLO) associated with apple proliferation was sequenced. Three putative open reading frames were observed on this fragment. The protein encoded by open reading frame 2 shows significant homologies with bacterial nitroreductases. From the nucleotide sequence four primer pairs for PCR were chosen to specifically amplify DNA from MLOs associated with European diseases of fruit trees. Primer pairs specific for (i) Malus-affecting MLOs, (ii) Malus- and Prunus-affecting MLOs, and (iii) Malus-, Prunus-, and Pyrus-affecting MLOs were obtained. Restriction enzyme analysis of the amplification products revealed restriction fragment length polymorphisms between Malus-, Prunus, and Pyrus-affecting MLOs as well as between different isolates of the apple proliferation MLO. No amplification with either primer pair could be obtained with DNA from 12 different MLOs experimentally maintained in periwinkle.     

New distal marker closely linked to the fragile X locus

Summary We have isolated II-10, a new X-chromosomal probe that identifies a highly informative two-allele TaqI restriction fragment length polymorphism at locus DXS466. Using somatic cell hybrids containing distinct portions of the long arm of the X chromosome, we could localize DXS466 between DXS296 and DXS304, both of which are closely linked distal markers for fragile X. This regional localization was supported by the analysis, in fragile X families, of recombination events between these three loci, the fragile X locus and locus DXS52, the latter being located at a more distal position. DXS466 is closely linked to the fragile X locus with a peak lod score of 7.79 at a recombination fraction of 0.02. Heterozygosity of DXS466 is approximately 50%. Its close proximity and relatively high informativity make DXS466 a valuable new diagnostic DNA marker for fragile X.     

Molecular Characterization of 16S Ribosomal DNA and Phylogenetic Analysis of Two X-disease Group Phytoplasmas Affecting China-tree (Melia azedarach L.) and Garlic (Allium sativum L.) in Argentina

Two phytoplasmas closely related to the X‐disease group were associated with China‐tree (Melia azedarach L.) and garlic (Allium sativum L.) decline diseases in Argentina. The present work was aimed at studying their phylogenetic relationship based on molecular characterization of the 16S ribosomal DNA sequences. Phytoplasma DNAs were obtained from naturally infected China‐tree and garlic plants from different geographical isolates. The results from analysis of restriction fragment length polymorphisms and nucleotide sequences of the 16S rDNA showed the affiliation of China‐tree and garlic decline phytoplasmas to the 16SrIII (X‐disease group), subgroups B and J, respectively. Both organisms had high sequence similarities in the 16SrDNA nucleotide sequence with the Chayote witches’ broom phytoplasma from Brazil. The phylogenetic tree, constructed by parsimony analysis, grouped the Garlic decline, China‐tree decline, Chayote witches’ broom and Clover yellow edge phytoplasmas into a cluster separated from the other phytoplasmas of the X‐disease group.     

Genetic variation at AFLPs for the Dipterocarpaceae and its relation to molecular phylogenies and taxonomic subdivisions

Genetic differentiation was investigated among 54 Indonesian species of Dipterocarpaceae, a dominant tree family in Asian tropical rainforests, using amplified fragment length polymorphism markers. The tree developed from the resultant unweighted pair group method using arithmetic averages clearly separated all investigated dipterocarps into two major groups that corresponded to tribe Dipterocarpeae and tribe Shoreae, respectively. These results are in accordance with the topology of molecular phylogenetic trees derived from PCR–restriction fragment length polymorphism analysis of chloroplast DNA and generally support the traditional taxonomic assessments. The possibility of interspecific hybridization is also discussed.     

Discrimination among the human beta A, beta S, and beta C-globin genes using allele-specific oligonucleotide hybridization probes.

Synthetic nonadecanucleotides complementary to the human beta A-, beta S-, or beta C-globin sequences were used as hybridization probes to screen human genomic DNA samples for these genes. The oligonucleotides were 32P-labeled and used as probes to genotype restriction endonuclease digests of human genomic DNA. The data obtained show that hybridization with oligonucleotide probes, unlike restriction fragment length polymorphism (RFLP) analysis or direct restriction enzyme digestion, can be used to directly distinguish among the three alleles of beta-globin, beta A, beta S, and beta C, when present either in one (heterozygous) or two copies.     

Human gamma X satellite DNA: an X chromosome specific centromeric DNA sequence

The cosmid clone, CX16-2D12, was previously localized to the centromeric region of the human X chromosome and shown to lack human X-specific satellite DNA. A 1.2 kb EcoRI fragment was subcloned from the CX16-2D12 cosmid and was named 2D12/E2. DNA sequencing revealed that this 1,205 bp fragment consisted of approximately five tandemly repeated DNA monomers of 220 bp. DNA sequence homology between the monomers of 2D12/E2 ranged from 72.8% to 78.6%. Interestingly, DNA sequence analysis of the 2D12/E2 clone displayed a change in monomer unit orientation between nucleotide positions 585–586 from a tail-to-head arrangement to a head-to-tail configuration. This may reflect the existence of at least one inversion within this repetitive DNA array in the centromeric region of the human X chromosome. The DNA consensus sequence derived from a compilation of these 220 bp monomers had approximately 62% DNA sequence similarity to the previously determined 8 satellite DNA consensus sequence. Comparison of the 2D12/E2 and 8 consensus sequences revealed a 20 bp DNA sequence that was well conserved in both DNA consensus sequences. Slot-blot analysis revealed that this repetitive DNA sequence comprises approximately 0.015% of the human genome, similar to that found with 8 satellite DNA. These observations suggest that this satellite DNA clone is derived from a subfamily of satellite DNA and is thus designated X satellite DNA. When genomic DNA from six unrelated males and two unrelated females was cut with SstI or HpaI and separated by pulsed-field gel electrophoresis, no restriction fragment length polymorphisms were observed for either X (2D12/E2) or 8 (50E4) probes. Fluorescence in situ hybridization localized the 2D12/E2 clone to the lateral sides of the primary constriction specifically on the human X chromosome.     

Genetic characterization of Legionella pneumophila serogroup 1 associated with respiratory disease in Australia.

Techniques were developed for genetic characterization of Legionella pneumophila serogroup 1 by using restriction fragment length polymorphism analysis. Allozyme analysis provided an index of the discrimination achieved by restriction fragment length polymorphism. Isolates from human cases of legionellosis were examined by both methods, and their profiles were compared with reference strains of L. pneumophila serogroup 1 obtained from the American Type Culture Collection. Eighteen distinct clones were evident among the isolates examined. Both methods could be used to trace the source of an outbreak of legionellosis caused by L. pneumophila serogroup 1.