RESUMEN
Elm yellows group (16SrV) phytoplasmas, which are associated with devastating diseases in elm, grapevine, blackberry, cherry, peach and several other plant species in America, Europe and Asia, represent one of the most diverse phytoplasma clusters. On the basis of phylogenetic analysis of 16S rDNA sequences, elm yellows group phytoplasmas form a discrete subclade within the phytoplasma clade. Three phylogenetic parameters, namely 16S rRNA, ribosomal protein and secY genes, have been evaluated for their usefulness in differentiating elm yellows group phytoplasmas. RFLP analysis of 16S rRNA sequences differentiated the elm yellows group phytoplasmas into five subgroups. Twelve RFLP subgroups were differentiated on the basis of ribosomal protein and 13 were differentiated using secY gene sequences. Phylogenetic analysis of the ribosomal protein genes and secY gene alone or in combination indicated that the subgroups constitute 12 genetically distinct lineages, each of which appears to have evolved under different ecological constraints such as specific vector or plant hosts. On the basis of unique DNA and biological properties, it is proposed that the elm yellows phytoplasma EY1(T) represents a novel taxon, 'Candidatus Phytoplasma ulmi'.
Asunto(s)
Acholeplasmataceae/clasificación , Ulmus/microbiología , Acholeplasmataceae/aislamiento & purificación , Acholeplasmataceae/patogenicidad , Secuencia de Bases , Cartilla de ADN , ADN Bacteriano/genética , ADN Ribosómico/genética , Datos de Secuencia Molecular , Filogenia , Enfermedades de las Plantas/microbiología , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , ARN Bacteriano/genética , ARN Ribosómico 16S/genéticaRESUMEN
Ribosomal (r) RNA interoperon sequence heterogeneity in the 'Fragaria multicipita' phytoplasma, a member of group 16SrVI, was initially observed in RFLP patterns of rDNA amplified in the polymerase chain reaction (PCR), and was confirmed through sequence analysis of cloned rDNA. Sequences from operons rrnA and rrnB were amplified in PCR primed by primer pair P1/P7 but from only rrnA in PCR primed by primer pair R16mF2/R16mR1. Preferential amplification of DNA from operon rrnA was explained by base mismatches between the R16mF2/R16mR1 primers and primer annealing sites in rrnB. The results revealed potential for classification of a phytoplasma into two different subgroups within a 16S rRNA group, if the phytoplasma's 16S rRNA gene sequences are independently characterized. It is suggested that the rRNA operon containing species-specific signature sequence(s) should be specified, and where possible sequences from both 16S rRNA genes should be included, in descriptions of new 'Candidatus Phytoplasma species'.
Asunto(s)
Acholeplasmataceae/clasificación , Acholeplasmataceae/genética , ADN Ribosómico , Polimorfismo de Longitud del Fragmento de Restricción , Operón de ARNr , Acholeplasmataceae/patogenicidad , ADN Bacteriano/genética , ADN Ribosómico/química , Fragaria/microbiología , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa , ARN Ribosómico 16S/genética , Mapeo Restrictivo , Análisis de Secuencia de ADN , Homología de SecuenciaRESUMEN
Phylogenetic relationships of five jujube witches'-broom (JWB) phytoplasma isolates from four different districts, and other phytoplasmas, were investigated by 16S rDNA PCR amplification and sequence analysis. The 16S rDNA sequences of any pair of the five isolates of JWB phytoplasmas were > 99.5% similar. The JWB phytoplasma 16S rDNA sequences were most closely related to that of the elm yellows (EY) phytoplasma in 16S-group VIII. Phylogenetic analysis of the 16S rDNA sequences from the JWB phytoplasma isolates, together with sequences from most of the phytoplasmas archived in GenBank, produced a tree in which the JWB isolates clustered as a discrete subgroup. The uniqueness of the JWB phytoplasma appears to be correlated with a specific insect vector (Hishimonus sellatus) and the host plant (Zizyphus jujuba), or with a specific geographical distribution. The unique properties of the JWB phytoplasma sequences clearly indicate that it represents a novel taxon, 'Candidatus Phytoplasma ziziphi'.
Asunto(s)
Acholeplasmataceae/clasificación , Enfermedades de las Plantas/microbiología , Ziziphus/microbiología , Acholeplasmataceae/genética , Acholeplasmataceae/aislamiento & purificación , Acholeplasmataceae/patogenicidad , Animales , Secuencia de Bases , ADN Bacteriano/genética , ADN Ribosómico/genética , Insectos Vectores/microbiología , Datos de Secuencia Molecular , Filogenia , ARN Bacteriano/genética , ARN Ribosómico 16S/genéticaRESUMEN
In Korea, Japanese chestnut trees (Castanea crenata Sieb. and Zucc.) showing symptoms indicative of witches' broom disease, including abnormally small leaves and yellowing of young leaves, were examined. Since the symptoms were suggestive of a phytoplasma infection, tissues were assayed for phytoplasmas by PCR analysis using a pair of universal primers that amplify a 1.4-kbp phytoplasma 16S rDNA fragment. The phytoplasma-specific fragment was amplified from diseased plants, but not from healthy plants, indicating that a phytoplasma was the causal agent of the chestnut witches' broom (CnWB) disease. The phylogenetic relationship of the CnWB phytoplasma to other phytoplasmas was examined by sequence analysis of the 16S rDNA. A phylogenetic analysis of 16S rDNA sequences of the phytoplasmas placed the CnWB phytoplasma within a distinct subgroup in the phytoplasma clade of the class Mollicutes. The phylogenetic tree indicated that the CnWB phytoplasma is related most closely to coconut phytoplasmas and suggested that they share a common ancestor. The unique properties of the CnWB phytoplasma sequences clearly establish that it represents a novel taxon, 'Candidatus Phytoplasma castaneae'.
Asunto(s)
Acholeplasmataceae/clasificación , Acholeplasmataceae/patogenicidad , Fagaceae/microbiología , Enfermedades de las Plantas/microbiología , Acholeplasmataceae/genética , Secuencia de Bases , Cocos/microbiología , ADN Bacteriano/genética , ADN Ribosómico/genética , Corea (Geográfico) , Datos de Secuencia Molecular , Filogenia , ARN Bacteriano/genética , ARN Ribosómico 16S/genéticaRESUMEN
Genes encoding SecA and SecY proteins, essential components of the Sec protein translocation system, were cloned from onion yellows phytoplasma, an unculturable plant pathogenic bacterium. The secA gene consists of 2,505 nucleotides encoding an 835 amino acid protein (95.7 kDa) and shows the highest similarity with SecA of Bacillus subtilis. Anti-SecA rabbit antibody was prepared from a purified partial SecA protein, with a histidine tag expressed in Escherichia coli. Western blot analysis confirmed that SecA protein (approximately 96 kDa) is produced in phytoplasma-infected plants. Immunohistochemical thin sections observed by optical microscopy showed that SecA is characteristically present in plant phloem tissues infected with phytoplasma. The secY gene consists of 1,239 nucleotides encoding a 413 amino acid protein (45.9 kDa) and shows the highest similarity with SecY of B. subtilis. These results suggest the presence of a functional Sec system in phytoplasmas. Because phytoplasmas are endocellular bacteria lacking cell walls, this system might secrete bacterial proteins directly into the host cytoplasm. This study is what we believe to be the first report of the sequence and expression analysis of phytoplasma genes encoding membrane proteins with a predicted function.
Asunto(s)
Acholeplasmataceae/genética , Adenosina Trifosfatasas/genética , Proteínas Bacterianas/genética , Proteínas de Escherichia coli , Genes Bacterianos , Proteínas de Transporte de Membrana/genética , Acholeplasmataceae/metabolismo , Acholeplasmataceae/patogenicidad , Adenosina Trifosfatasas/inmunología , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Animales , Asteraceae/microbiología , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Clonación Molecular , ADN Bacteriano/genética , Expresión Génica , Inmunohistoquímica , Proteínas de Transporte de Membrana/inmunología , Proteínas de Transporte de Membrana/metabolismo , Datos de Secuencia Molecular , Mapeo Físico de Cromosoma , Conejos , Canales de Translocación SEC , Proteína SecA , Homología de Secuencia de AminoácidoRESUMEN
Hibiscus rosa-sinensis is a valuable ornamental species widely planted in Brazil. Many plants are affected by witches' broom disease, which is characterized by excessive axillary branching, abnormally small leaves, and deformed flowers, symptoms that are characteristic of diseases attributed to phytoplasmas. A phytoplasma was detected in diseased Hibiscus by amplification of rRNA operon sequences by PCRs, and was characterized by RFLP and nucleotide sequence analyses of 16S rDNA. The collective RFLP patterns of amplified 16S rDNA differed from the patterns described previously for other phytoplasmas. On the basis of the RFLP patterns, the hibiscus witches' broom phytoplasma was classified in a new 16S rRNA RFLP group, designated group 16SrXV. A phylogenetic analysis of 16S rDNA sequences from this and other phytoplasmas identified the hibiscus witches' broom phytoplasma as a member of a distinct subclade (designated subclade xiv) of the class Mollicutes. A phylogenetic tree constructed on the basis of 16S rRNA gene sequences was consistent with the hypothesis that there was divergent evolution of hibiscus witches' broom phytoplasma and its closest relatives (members of 16S rRNA RFLP group 16SrII) from a common ancestor. On the basis of unique properties of the DNA from hibiscus witches' broom phytoplasma, it is proposed that it represents a new taxon, namely 'Candidatus Phytoplasma brasiliense'.
Asunto(s)
Acholeplasmataceae/clasificación , Filogenia , Rosales/microbiología , Acholeplasmataceae/genética , Acholeplasmataceae/patogenicidad , ADN Ribosómico/genética , Datos de Secuencia Molecular , Enfermedades de las Plantas/microbiología , Hojas de la Planta , Tallos de la Planta , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , ARN Ribosómico 16S/genética , Mapeo RestrictivoRESUMEN
In terms of biosystematics, the plant-pathogenic mycoplasmalike organisms (MLOs) have been tentatively placed into the class Mollicutes. Certain physiological tests have been used to distinguish families within this class: the sterol-nonrequiring Acholeplasmataceae differ from the sterol-requiring Mycoplasmataceae in that the former are more resistant to lysis by digitonin and more sensitive to lysis in hypotonic salt solutions. To test MLOs for these membrane properties and thus assist in their definitive classification, a dot-blot microassay procedure was used to detect nucleic acids released from lysed cells. The results show that MLOs resemble acholeplasmas grown in the absence of sterols in that they are resistant to digitonin and sensitive to hypotonic salt solutions. The MLOs can be differentiated from acholeplasmas grown without sterols by their greater resistance to lysis in hypotonic sucrose solutions.