Odile Domergue

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.

Multilocus sequence analysis of root nodule isolates from Lotus arabicus (Senegal), Lotus creticus , Argyrolobium uniflorum and Medicago sativa (Tunisia) and description of Ensifer numidicus sp nov and Ensifer garamanticus sp. nov.

Nine isolates from Argyrolobium uniflorum, Lotus creticus , Medicago sativa (Tunisia) and Lotus arabicus (Senegal) were analysed by multilocus sequence analysis (MLSA) of five housekeeping genes (recA, atpD, glnA, gltA and thrC), the 16S rRNA gene and the nodulation gene nodA. Analysis of the individual and concatenated gene sequences demonstrated that the nine new strains constituted three stable, well-supported (bootstrap and gene sequence similarity values) monophyletic clusters, A, B and C, all belonging to the branch of the genus Ensifer, regardless of the phylogenetic reconstruction method used (maximum likelihood, maximum-parsimony, neighbour-joining). The three groups were further characterized by API 100 auxanographic tests, host specificity and nodA gene sequence analysis. On the basis of these data, clusters A and C are suggested as representing two novel species within the genus Ensifer, for which the names Ensifer numidicus sp. nov. (type strain ORS 1407(T)=LMG 24690(T)=CIP 109850(T)) and Ensifer garamanticus sp. nov. (type strain ORS 1400(T)=LMG 24692(T)=CIP 109916(T)) are proposed. The cluster B strains were assigned to Ensifer adhaerens genomovar A.

Symbiotic characterization and diversity of rhizobia associated with native and introduced acacias in arid and semi-arid regions in Algeria.

The diversity of rhizobia associated with introduced and native Acacia species in Algeria was investigated from soil samples collected across seven districts distributed in arid and semi-arid zones. The in vitro tolerances of rhizobial strains to NaCl and high temperature in pure culture varied greatly regardless of their geographical and host plant origins but were not correlated with the corresponding edaphoclimatic characteristics of the sampling sites, as clearly demonstrated by principal component analysis. Based on 16S rRNA gene sequence comparisons, the 48 new strains isolated were ranked into 10 phylogenetic groups representing five bacterial genera, namely, Ensifer, Mesorhizobium, Rhizobium, Bradyrhizobium, and Ochrobactrum. Acacia saligna, an introduced species, appeared as the most promiscuous host because it was efficiently nodulated with the widest diversity of rhizobia taxa including both fast-growing ones, Rhizobium, Ensifer, and Mesorhizobium, and slow-growing Bradyrhizobium. The five other Acacia species studied were associated with fast-growing bacterial taxa exclusively. No difference in efficiency was found between bacterial taxa isolated from a given Acacia species. The tolerances of strains to salinity and temperature remains to be tested in symbiosis with their host plants to select the most adapted Acacia sp.-LNB taxa associations for further revegetation programs.

Bradyrhizobia Nodulating the Acacia mangium × A. auriculiformis Interspecific Hybrid Are Specific and Differ from Those Associated with Both Parental Species

ABSTRACT In the context of an increasing utilization of the interspecific hybrid Acacia mangium × A. auriculiformis as a plantation tree in the tropical humid zone, its symbiotic characterization was carried out in comparison with that of its two parental species. Rhizobium strains of diverse geographical origins were isolated from root nodules of the hybrid and its parents. Almost all Acacia hybrid isolates were fast growing on yeast extract-mannitol medium, in contrast to those isolated from both parental species, which were mostly slow growing. The rhizobium strains were characterized through partial sequencing of the rRNA operon. In the phylogenetic tree, almost all strains isolated from the hybrid were grouped together in a clade close to Bradyrhizobium japonicum, while all strains isolated from both parental species were close to Bradyrhizobium elkanii. Inoculation experiments performed under in vitro or greenhouse conditions showed that all strains were infective with their original hosts but exhibited very variable degrees of effectivity according to the host plant tested. Thus, homologous strain-host associations were more effective than heterologous ones. This shows that there is still a high potential for isolating and testing new strains from hybrids to be used as inoculants in the context of large-scale afforestation programs.

Distribution of Medicago Species and Their Microsymbionts in a Saline Region of Algeria

We studied symbiosis of Medicago ciliaris and Medicago polymorpha, two legumes of forage and ecological importance in Algeria, especially in saline soil regions. We report the spatial distribution of the two species and their microsymbionts along salinity gradient transects in the Sebkha of Misserghin (Algeria, North Africa). Seeds and root nodules were sampled from 10 sites. Twenty-seven rhizobial strains were isolated from root nodules and characterized as fast-growers and slime-producers on yeast mannitol agar. By partial sequencing of the gene coding for the 16 S ribosomal RNA, they were found to be affiliated to Rhizobium, Sinorhizobium, and Agrobacterium but several strains had sequences with separate positions.Interestingly one of these was further assigned to Phyllobacterium. Opposite to rhizobia, the distribution of the two Medicago species varied along the salinity gradient, M. ciliaris being dominant in the low NaCl concentration zones and M. polymorpha dominant in the most saline zones. Tolerance to salinity, of both bacterial and plant partners, was studied under laboratory conditions, showing that plants are susceptible to NaCl concentrations of 50 mM, 15-fold lower than that of their associated rhizobia (800 mM).

Nodular bacterial endophyte diversity associated with native Acacia spp. in desert region of Algeria

Five species of Acacia (Acacia ehrenbergiana Hayne, A. nilotica (L.) Delile, A. seyal Delile, A. tortilis (Forssk.) Hayne and A. laeta Delile) indigenous to Tamanrasset (Algeria) were investigated for their nodulation status and nodular endophytic diversity. A. ehrenbergiana showed the highest nodulation ability across the different sites in this region, indicating the widespread occurrence of compatible rhizobia in the soils. Altogether 81 strains were purified. Among this endophytic strain collection, only four bacterial endophytes nodulated their respective host plants. On the basis of partial 16S rDNA sequencing, they were affiliated to Ensifer sp., Ensifer teranga, Mesorhizobium sp. and Rhizobium sp. Among the 79 non- symbiotic endophytes, 24 representative strains on the basis of PCR-RFLP profile obtained with MSPI enzyme digestion were characterized. They belonged to nine genera, namely: Paenibacillus, Ochrobactrum, Stenotrophomonas, Pseudomonas, Microbacterium, Rhizobium, Agrobacterium, Brevibacillus and Advenella. The isolated nodular endophytes in this study revealed a strong tolerance profile to salinity and high temperature. Principal component analysis confirmed that no correlation was found between bacterial tolerance to a maximum temperature of growth and soil depth of sampling. This tolerance profile was distributed over the three levels of soil depth sampling: 20, 40 and 60 cm. On the other hand, there was no relationship between in vitro tolerances of rhizobial strains to NaCl and high temperature and corresponding edaphoclimatic characteristics of the sampling sites. This study is a contribution to nodular bacterial diversity knowledge of desert African Acacia species growing in preserved ecosystems. Key words: Acacia spp., bacterial endophytic diversity, desert, nodules, salinity tolerance, temperature tolerance.