[Modern bacterial taxonomy: techniques review--application to bacteria that nodulate leguminous plants (BNL)]. / La taxonomie bactérienne moderne: revue des techniques--application à la caractérisation des bactéries nodulant les légumineuses (BNL).

Taxonomy is the science that studies the relationships between organisms. It comprises classification, nomenclature, and identification. Modern bacterial taxonomy is polyphasic. This means that it is based on several molecular techniques, each one retrieving the information at different cellular levels (proteins, fatty acids, DNA...). The obtained results are combined and analysed to reach a "consensus taxonomy" of a microorganism. Until 1970, a small number of classification techniques were available for microbiologists (mainly phenotypic characterization was performed: a legume species nodulation ability for a Rhizobium, for example). With the development of techniques based on polymerase chain reaction for characterization, the bacterial taxonomy has undergone great changes. In particular, the classification of the legume nodulating bacteria has been repeatedly modified over the last 20 years. We present here a review of the currently used molecular techniques in bacterial characterization, with examples of application of these techniques for the study of the legume nodulating bacteria.

Diverse bacteria associated with root nodules of spontaneous legumes in Tunisia and first report for nifH-like gene within the genera Microbacterium and Starkeya.

We characterized 34 endophytic bacterial isolates associated to root nodules collected from spontaneous legumes in the arid zone of Tunisia by 16S rDNA polymerase chain reaction (PCR)-restriction fragment length polymorphism, whole cell protein sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), 16S rDNA and 16S-23S rDNA internal transcribed spacer sequencing. Phylogenetically, these isolates belong to the branches containing the genera Inquilinus, Bosea, Rhodopseudomonas, Paracraurococcus, Phyllobacterium, Ochrobactrum, Starkeya, Sphingomonas, Pseudomonas, Agromyces, Microbacterium, Ornithinicoccus, Bacillus, and Paenibacillus. These strains did not induce any nodule formation when inoculated on the wide host spectrum legume species M. atropurpureum (Siratro) and no nodA gene could be amplified by PCR. However, nifH sequences, most similar to those of Sinorhizobium meliloti, were detected within strains related to the genera Microbacterium, Agromyces, Starkeya and Phyllobacterium.

Emended description of the genus Phyllobacterium and description of four novel species associated with plant roots: Phyllobacterium bourgognense sp. nov., Phyllobacterium ifriqiyense sp. nov., Phyllobacterium leguminum sp. nov. and Phyllobacterium brassicacearum sp. nov.

Gram-negative bacteria were isolated from the rhizoplane of Brassica napus in France and from root nodules of Argyrolobium uniflorum, Astragalus algerianus and Lathyrus numidicus growing in the infra-arid zone of southern Tunisia. Based on phylogenetic analysis of the 16S rRNA gene sequences, the seven isolates belong to the Alphaproteobacteria and are related to Phyllobacterium myrsinacearum strains. The isolates formed three clusters; clusters A and C consist of Tunisian strains, whereas cluster B consists of two strains from Brassica napus from France. Phylogenetic reconstruction based on the atpD gene strongly supports their affiliation to the genus Phyllobacterium. DNA-DNA hybridizations revealed that (i) none of the isolates belong to the species P. myrsinacearum, (ii) clusters A and C represent two distinct genomospecies and (iii) the two strains of cluster B represent two separate genomospecies. Distinctive phenotypic features were deduced from numerical analysis of phenotypic data. Based on this polyphasic approach, four novel species are proposed: Phyllobacterium leguminum sp. nov. (type strain ORS 1419T = CFBP 6745T = LMG 22833T), Phyllobacterium ifriqiyense sp. nov. (type strain STM 370T = CFBP 6742T = LMG 22831T), Phyllobacterium brassicacearum sp. nov. (type strain STM 196T = CFBP 5551T = LMG 22836T) and Phyllobacterium bourgognense sp. nov. (type strain STM 201T = CFBP 5553T = LMG 22837T). The description of the genus Phyllobacterium is emended accordingly.

Characterisation of wild legume nodulating bacteria (LNB) in the infra-arid zone of Tunisia.

We report on the isolation and the characterization of nitrogen-fixing root nodule bacteria isolated from natural legumes in a region of South Tunisia corresponding to the infra-arid climatic zone. A collection of 60 new bacterial root nodule isolates were obtained from 19 legume species belonging to the genera Acacia, Anthyllis, Argyrolobium, Astragalus, Calycotome, Coronilla, Ebenus, Genista, Hedysarum, Hippocrepis, Lathyrus, Lotus, Medicago, Ononis. The isolates were characterised by (1) comparative 16S ARDRA using 7 enzymes, (2) total cell protein SDS-PAGE analysis and (3) 16S rDNA sequencing. The results show that these isolates are diverse and belong to the genera Rhizobium, Sinorhizobium, Mesorhizobium and Bradyrhizobium. Bradyrhizobium were further characterised by 16S-23S rDNA IGS sequencing. Surprisingly strains nodulating Astragalus cruciatus, Lotus creticus and Anthyllis henoniana were identified as Rhizobium galegae, a species recorded only as endosymbiont of Galega officinalis and G. orientalis in northern regions so far.