RESUMO
An optimized technique of polyacrylamide gel electrophoresis, Staircase Electrophoresis (SCE), was applied to determine the stable Low Molecular Weight RNA (LMW RNA) profiles of 25 Frankia strains from diverse geographic origins and host specificity groups as well as species from other actinomycete genera. Application of the technique permits the rapid identification of Frankia strains and their differentiation from other actinomycetes. The isolates used in this study were grouped in eight clusters, each comprising strains with identical LMW RNA profiles. Comparison of these results with others obtained from DNA sequences or DNA hybridization methods suggest a high degree of complexity in the genus Frankia. Application of SCE to profile LMW RNA should in the future facilitate biodiversity studies of Frankia and discrimination of new species.
Assuntos
Actinomycetales/classificação , Actinomycetales/genética , Eletroforese em Gel de Poliacrilamida/métodos , RNA Bacteriano/análise , Peso Molecular , Plantas/microbiologiaRESUMO
Among the Frankia strains capable of establishing a nitrogen-fixing symbiosis with actinorrhizal plants, in planta sporangial formation is not universal and has led to the distinction between spore-positive (Sp+) and spore-negative (Sp-) nodules. Numerous Frankia strains have been isolated in pure culture from Sp+ nodules of different host plants, but, although they were able to reinfect their respective host plant, none of them was able to differentiate endophytic sporangia under laboratory conditions. The first step of this study was to demonstrate, at the molecular level, the existence of specific Sp+ strain genotypes differing from Sp- strain genotypes in a single alder stand. In a second step, by way of PCR amplification and sequencing of the PCR products, we have characterized oligonucleotide primers specific for the genus Frankia and for each of the two types of Frankia microsymbionts able, or not, to differentiate sporangia inside natural green alder nodules. These primers applied in PCRs with DNA extracted from nodules confirmed the morphological identification and revealed the presence of nodules colonized by both types of actinomycetes. Finally, a preliminary PCR study was conducted with DNA extracted directly from soil samples which permitted checking the rhizosphere of Sp+ and Sp- nodules for the presence of the corresponding strains.
Assuntos
Actinomycetales/isolamento & purificação , Fixação de Nitrogênio , Microbiologia do Solo , Árvores/microbiologia , Actinomycetales/genética , Actinomycetales/fisiologia , Sequência de Bases , DNA Bacteriano/análise , Genótipo , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Esporos Bacterianos , SimbioseRESUMO
Repeated attempts at isolating the Frankia endophyte of Coriaria spp. have not yielded infective microbial cultures that could fulfil Koch's postulates. In order to circumvent the critical isolation step, nodule endophytes of Coriaria were characterized directly by means of specific amplification of nodule DNA (PCR) followed by sequencing of part of the 16S rDNA gene. Three closely related sequences were obtained from nodules originating from France, Mexico and New Zealand, containing unique sequences different from all other Frankia strains characterized so far. The sequences obtained were closest (with 5 or 6 substitutions) to those of Frankia alni and those of Casuarina-infective Frankia strains, respectively. Two nucleotides unique to the Coriaria endophyte sequences were used to define specific primers, resulting in a hybridization test that could discriminate between Frankia DNAs originating from Coriaria nodules and those recovered from all cultured Frankia strains tested. The endophytes of Coriaria thus appear to form a distinct Frankia lineage.
Assuntos
Actinomycetales/genética , Actinomycetales/isolamento & purificação , DNA Fúngico/genética , Plantas/microbiologia , Actinomycetales/classificação , Sequência de Bases , Primers do DNA/genética , DNA Fúngico/isolamento & purificação , DNA Ribossômico/genética , Ecossistema , Variação Genética , Dados de Sequência Molecular , Filogenia , Plantas/genética , SimbioseRESUMO
A set of oligonucleotides has been developed to study the competitivity of two Frankia strains in the nodulation of the roots of two host plant species: Alnus glutinosa and Alnus incana. Two 20 mer-oligonucleotides, complementary to highly conserved sequences inside the nifH gene, were used as primers for the polymerase chain reaction (PCR) system in order to amplify microsymbiont DNA extracted from actinorhizae. PCR products were analyzed using two strain-specific 15-mer oligonucleotides identified in the amplified region. Hybridization data indicate that strain ACoN24d is more competitive than strain ArI3 in the nodulation of both hosts.
Assuntos
Actinomycetales/isolamento & purificação , DNA Bacteriano/análise , Hibridização de Ácido Nucleico , Sondas de Oligonucleotídeos , Actinomycetales/genética , Sequência de Bases , Southern Blotting , DNA Bacteriano/genética , Dados de Sequência Molecular , Plantas , Reação em Cadeia da Polimerase , Especificidade da EspécieRESUMO
A method to achieve cell lysis and isolate Frankia sp. plasmid DNA was developed. A screening of Frankia sp. strains belonging to different host compatibility groups (Alnus sp., Elaeagnus sp., Ceanothus sp.) showed that, of 39 strains tested, 4 (strains Cp11, ARgN22d, ArI3, and EUN1f) possessed plasmids ranging in size from 7.1 to 32.2 kilobase pairs as estimated from agarose gel electrophoresis and electron microscopy. A total of 11 plasmids were detected.
