Mosbah Mahdhi

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.

Paenibacillus prosopidis sp. nov., isolated from the nodules of Prosopis farcta

A bacterial strain, designated PW21(T), was isolated from root nodules of Prosopis farcta in Tunisia. Phylogenetic analysis based on 16S rRNA gene sequences placed the isolate into the genus Paenibacillus, with its closest relatives being Paenibacillus glycanilyticus DS-1(T) and Paenibacillus castaneae Ch-32(T) with identity values of 96.9 %. DNA-DNA hybridization measurements showed values of less than 25 % with respect to these two species. The isolate was a Gram-variable, motile and sporulating rod. Catalase activity was positive and oxidase activity was weakly positive. Aesculin, CM-cellulose, xylan and starch were hydrolysed but casein and gelatin were not. Acetoin production was weakly positive and nitrate reduction was negative. Urease production was negative. Growth was supported by many carbohydrates and organic acids as carbon sources. MK-7 was the predominant menaquinone and anteiso-C(15 : 0), iso-C(16 : 0) and iso-C(15 : 0) were the major fatty acids. Major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, a glycolipid, six phospholipids, an unidentified lipid and two unknown aminophosphoglycolipids. meso-Diaminopimelic acid was not detected in the peptidoglycan. The DNA G+C content of the isolate was 52.9 mol%. Phylogenetic, chemotaxonomic and phenotypic analyses showed that strain PW21(T) should be considered to represent a novel species of the genus Paenibacillus, for which the name Paenibacillus prosopidis sp. nov. is proposed. The type strain is PW21(T) (=LMG 25259(T) =CECT 7506(T) =DSM 22405(T)).

Phenotypic and genotypic diversity of Genista saharae microsymbionts from the infra-arid region of Tunisia.

Aims:  Genista saharae, indigenous of Sahara, is a spontaneous shrub that plays an important ecological role for the preservation and fertility of poor and eroded soils. This legume has not been examined for its root nodule bacteria. The taxonomic diversity of bacteria from root nodules of G. saharae growing in the infra‐arid region of Tunisia was investigated.

Genotypic diversity of rhizobia isolated fromRetama raetam in arid regions of Tunisia

Thirty-five isolates of rhizobia were picked up fromRetama raetam root nodules growing in arid lands of Tunisia. A genotypic approach including PCR-RFLP of 16S rDNA and 16S–23S rDNA was used to study their diversity and their relationships with te n reference strains of rhizobia. Four distinct clusters were defined in numerical analysis of RFLP of 16S rDNA, which related at the 78% similarity level to distinct species ofMesorhizobium, Agrobacterium, Rhizobium andSinorhizobium. More greater variability was detected by analysis of Intergenic Spacers 16S–23S rDNA. The results from both methods used in this study, showed that among all newsolates only three were found to be closely related to species of the genusSinorhizobium.

Characterization of root-nodulating bacteria associated to Prosopis farcta growing in the arid regions of Tunisia.

Diversity of 50 bacterial isolates recovered from root nodules of Prosopis farcta grown in different arid soils in Tunisia, was investigated. Characterization of isolates was assessed using a polyphasic approach including phenotypic characteristics, 16S rRNA gene PCR–RFLP and sequencing, nodA gene sequencing and MLSA. It was found that most of isolates are tolerant to high temperature (40°C) and salinity (3%). Genetic characterization emphasizes that isolates were assigned to the genus Ensifer (80%), Mesorhizobium (4%) and non-nodulating endophytic bacteria (16%). Forty isolates belonging to the genus Ensifer were affiliated to Ensifer meliloti, Ensifer xinjiangense/Ensifer fredii and Ensifer numidicus species. Two isolates belonged to the genus Mesorhizobium. Eight isolates failing to renodulate their host plant were endophytic bacteria and belonged to Bacillus, Paenibacillus and Acinetobacter genera. Symbiotic properties of nodulating isolates showed a diversity in their capacity to infect their host plant and fix atmospheric nitrogen. Isolate PG29 identified as Ensifer meliloti was the most effective one. Ability of Prosopis farcta to establish symbiosis with rhizobial species confers an important advantage for this species to be used in reforestation programs. This study offered the first systematic information about the diversity of microsymbionts nodulating Prosopis farcta in the arid regions of Tunisia.

Legume-nodulating bacteria (LNB) from three pasture legumes ( Vicia sativa , Trigonella maritima and Hedysarum spinosissimum ) in Tunisia

Sixty-one bacterial isolates were recovered from surface-sterilized root nodules of Vicia sativa, Trigonella maritima and Hedysarum spinosissimum plants growing in two arid Tunisian soils. The natural nodulation resource of these legumes, prospected from the two sites, was investigated. The occurrence of nodulation and the morphology of the nodules were observed. The isolates were examined by phenotypic characterization and 16S rDNA analysis. Among the 61 isolates that were screened, the majority (92%) were fast-growing rhizobia. Twenty-eight strains tolerated high concentration of salt (3% NaCl) and grew at temperatures up to 40°C. PCR restriction fragment length polymorphism (PCR-RFLP) and 16S rRNA gene sequencing revealed that the majority of the isolates belonged to the genera Rhizobium (54%) and Sinorhizobium (42%). Five H. spinosissimum isolates failed to nodulate their host plant, and were affiliated to Pseudomonas and Kocuria genera. This study is the first report that describes bacteria of genus Kocuria occupying root nodules of legumes to the best of our knowledge.

Taxonomic and symbiotic diversity of bacteria isolated from nodules of Acacia tortilis subsp. raddiana in arid soils of Tunisia.

A collection of rhizobia isolated from Acacia tortilis subsp. raddiana nodules from various arid soils in Tunisia was analyzed for their diversity at both taxonomic and symbiotic levels. The isolates were found to be phenotypically diverse. The majority of the isolates tolerated 3% NaCl and grew at 40 °C. Genetic characterization emphasized that most of the strains (42/50) belong to the genus Ensifer, particularly the species Ensifer meliloti, Ensifer garamanticus, and Ensifer numidicus. Symbiotic properties of isolates showed diversity in their capacity to nodulate their host plant and to fix atmospheric nitrogen. The most effective isolates were closely related to E. garamanticus. Nodulation tests showed that 3 strains belonging to Mesorhizobium genus failed to renodulate their host plant, which is surprising for symbiotic rhizobia. Furthermore, our results support the presence of non-nodulating endophytic bacteria belonging to the Acinetobacter genus in legume nodules.

Seasonal Changes of Microbiological Properties in Steppe Soils from Degraded Arid Area in Tunisia

There is growing interest in assessing soil quality using microbial properties in desertified areas. A study was conducted in arid soils subjected to desertification in the south of Tunisia to illustrate the effects of dominating steppes of Stipa tenacissima and Anthyllis sericea subsp. henoniana on soil chemical, microbial, and biochemical properties. Soil samples were collected beneath the canopy of S. tenacissima, A. sericea subsp. Henoniana, and open areas in the rainy and dry seasons. These steppes showed higher values of soil organic carbon content (Corg). Microbial biomass carbon (Cmic) was also greater under steppes and reached 289 μg C g−1 soil under Anthyllis canopies. Studied enzyme activities (dehydrogenase, phosphatase and β-glucosidase) increased greatly beneath steppes showing dehydrogenase activity of 159 μg INTF g−1 soil d−1, while it did not reach 36 μg INTF g−1 soil d−1 in open areas. Conversely, lower values of C/N ratio and metabolic quotient (qCO2), particularly in the vicinity of A. sericea subsp. henoniana. The environmental conditions significantly affected studied properties: the soil organic carbon, microbial biomass, and enzyme activities were reduced in dry season, while C/N ratio and qCO2 were higher. Our results suggest the importance of vegetation cover in regulating soil microbial processes in arid degraded areas and that the wet season may play an important role in driving seasonal changes in microbial biomass and activity.

Characterization of Rhizobial Bacteria Nodulating Astragalus corrugatus and Hippocrepis areolata in Tunisian Arid Soils

Fifty seven bacterial isolates from root nodules of two spontaneous legumes (Astragalus corrugatus and Hippocrepis areolata) growing in the arid areas of Tunisia were characterized by phenotypic features, 16S rDNA PCR-RFLP and 16S rRNA gene sequencing. Phenotypically, our results indicate that A. corrugatus and H. areolata isolates showed heterogenic responses to the different phenotypic features. All isolates were acid producers, fast growers and all of them used different compounds as sole carbon and nitrogen source. The majority of isolate grew at pHs between 6 and 9, at temperatures up to 40°C and tolerated 3% NaCl concentrations. Phylogenetically, the new isolates were affiliated to four genera Sinorhizobium, Rhizobium, Mesorhizobium and Agrobacterium. About 73% of the isolates were species within the genera Sinorhizobium and Rhizobium. The isolates which failed to nodulate their host plants of origin were associated to Agrobacterium genus (three isolates).