Silvia Fancelli

Genetic Diversity and Dynamics of Sinorhizobium meliloti Populations Nodulating Different Alfalfa Cultivars in Italian Soils

ABSTRACT We analyzed the genetic diversity of 531 Sinorhizobium meliloti strains isolated from nodules of Medicago sativa cultivars in two different Italian soils during 4 years of plant growth. The isolates were analyzed for DNA polymorphism with the random amplified polymorphic DNA method. The populations showed a high level of genetic polymorphism distributed throughout all the isolates, with 440 different haplotypes. Analysis of molecular variance allowed us to relate the genetic structure of the symbiotic population to various factors, including soil type, alfalfa cultivar, individual plants within a cultivar, and time. Some of these factors significantly affected the genetic structure of the population, and their relative influence changed with time. At the beginning of the experiment, the soil of origin and, even more, the cultivar significantly influenced the distribution of genetic variability of S. meliloti. After 3 years, the rhizobium population was altered; it showed a genetic structure based mainly on differences among plants, while the effects of soil and cultivar were not significant.

Influence of plant genotype on the selection of nodulating Sinorhizobium meliloti strains by Medicago sativa

We analysed the genetic diversity of 270 Sinorhizobium meliloti strains isolated from nodules of three different Medicago sativa varieties, planted in three different Italian soils, combining the Analysis of Molecular Variance (AMOVA) with the Random Amplified Polymorphic DNA (RAPD) technique to estimate variance among RAPD patterns with the aim to draw an objective description of the population genetic structure. Results indicated that a general intraspecific genetic diversity was globally distributed among all the population, however a very high level of diversity was found among strains nodulating different Medicago sativa varieties. Moreover the distribution of the RAPD haplotypes among the plant varieties also showed to be non-random. The overall data indicated that the plant genotype is a major factor in shaping the genetic structure of this natural Rhizobium population.

Molecular characterization of rhizosphere and clinical isolates of Burkholderia cepacia

Four Burkholderia cepacia strains isolated from the rhizosphere and pathological samples of infected human patients were characterized at the molecular level by different methodologies, including the determination of 16S ribosomal rDNA sequence, restriction endonuclease analysis of total DNA, random amplified polymorphic DNA fingerprinting and Southern hybridization with gene probes for nitrogen fixation and siderophore synthesis. The results indicate that the four strains cluster together within genus Burkholderia, but differ from one another. The DNA from the four strains hybridized to the nifA gene probe from Klebsiella pneumoniae, and an appreciable homology with the nifHDK structural genes of Azospirillum brasilense was demonstrated for one rhizosphere strain. Although the four isolates produced an ornibactin-like siderophore, they did not give hybridization with the pvdA probe for hydroxamate biosynthesis from Pseudomonas aeruginosa.

Phylogenetic Studies of the Genus Azospirillum

The 16S rDNA of seventeen Azospirillum strains, fourteen of which assigned to one of known A. amazonense, A. brasilense, A. halopraeferens, A. irakense and A. lipoferum species, and other three of uncertain taxonomic collocation, was sequenced and analysed after polymerase chain reaction amplification, in order to investigate the phylogenetic relationships at the intra-generic and super-generic level. Moreover the use of histidine biosynthetic genes and of anonymous sequences generated by RAPD technique to infer phylogenetic relationships among Azospirillum strains is also described. The relationships among the five species, as revealed by DNA sequence data, appeared to be congruent with other molecular and phenotypic results, showing that the genus Azospirillum constitutes a phylogenetically separate entity in which A. brasilense and A. lipoferum are very close species, whereas the species A. amazonense is the most divergent.

Heterologous gene expression in an Escherichia coli population under starvation stress conditions

Abstract. A novel system to study the evolution of transcription signals in heterologous systems under selective starvation conditions is described. It is based on the plasmid-mediated transfer of his biosynthetic genes from Azospirillum brasilense into a heterologous Escherichia coli mutant population lacking histidine biosynthetic ability. We show that under highly selective stressful conditions, genetic changes in the donor plasmid lead to mutated sequences that are efficiently recognized as promoters by the E. coli RNA polymerase.

Phylogeny of the genus based on 16S rDNA sequence

The 16S rDNA of 17 strains of Azospirillum, 14 assigned to one of the known species A. amazonense A. brasilense A. halopraeferens A. irakense and A. lipoferum, and the other three of uncertain taxonomic position, was sequenced after polymerase chain reaction amplification and analysed in order to investigate the phylogenetic relationships at the intra-generic and super-generic level. The phylogenetic analysis confirms that the genus Azospirillum constitutes a phylogenetically separate entity within the a subclass of Proteobacteria and that the five species are well defined. A. brasilense and A. lipoferum are closely related species and form one cluster together with A. halopraeferens; the pair of species A. amazonense and A. irakense forms a second cluster in which Rhodospirillum centenum is also placed.