Caractérisation des isolats bactériens par des techniques phénotyques et électrophorétiques isolés à partir des nodules de queques espèces de légumineuses spontanées de la tribu des Ginesteae (Fabaceae)

Abstract

Most studies of rhizobia have focused on identification and systematics of symbionts nodulating crop legumes because of their interest in improving yield. However, few studies investigated the endosymbiotic nitrogen-fixing bacteria nodulating wild and spontaneous legume species, in particular in North Africa. This study aims to phenotypically characterize endosymbiotic bacteria isolated from root-nodules of a wild legumes growing in arid and semiarid regions of northeast Algeria. Genista microcephala and Argyrolobium uniflorum by chemotaxonomic, biochemical profiling and symbiotic properties comparing them with reference strains (RS). In addition polyphasic characterization of bacteria isolated from nodules of Genista cinerea. It examines growth variations of endosymbiont isolates under different environmental conditions including severe situations and to determine their taxonomic position based on growth tolerance limits and potential adaptations, using advanced statistical modelling techniques and genomic characterization based on 16S rRNA sequencing. Phenotypic characterization was performed using physiological tests (different pH levels, NaCl concentrations, and tolerance to temperature), nutritional experiments (assimilation of different carbohydrates and amino acids) and biochemical profiling (antibiotic resistance, heavy metals resistance and protein profile). Polyphasic characterization was conducted using physiological tests, nutritional experiments and genomic characterization based on 16S rRNA sequencing. Data were analyzed using Markov Chain Monte Carlo sampler for multivariate generalized linear mixed models (MCMCglmm) to detect growth differences between isolates and RS. Similarities between isolates and RS were assessed using agglomerative hierarchical clustering (AHC), whereas multiple factor analysis (MFA) was performed to understand factors influencing each group of isolates/RS and Phylogenie of isolates was conducted. The results showed that the isolates can grow at pH [4–10], salt concentration [NaCl=0–5%] and temperature up to 45°C. The rhizobia associated with G. microcephala and A. uniflorum were able to produce different hydrolytic enzymes including cellulose, pectinase and urease, with remarkable tolerance to toxic metals such as zinc, lead, copper, and mercury. Numerical analysis of the phenotypic characteristics revealed that the rhizobial isolates formed four main distinct groups showing high levels of similarity with Gammaproteobacteria. Symbiotic and cultural characteristics of G. cinerea isolats revealed the existence of a large physiological diversity among tested bacteria, which showed a broad capability to assimilate different carbonaceous and nitrogenous substances, with consistent and large tolerances to pH [4–10], temperature [4–55°C], and salinity [NaCl=2–10%]. Although, the endosymbiont isolates have broad metabolic diversity, they formed two distinct groups with high level of similarity with RS. Group 1 included fast-growing and salt-tolerant isolates characterized by tolerance to acidity with high growth in alkaline conditions. Group 2 covered slow-growing acid-sensitive isolates that high salinity negatively affected their growth. Results of AHC and MFA evidenced that bacterial diversity of endosymbiont isolates showed high level of similarity with RS, a proof that they are rhizobial strains. In concordance with phylogenitic results isolates are closely related to different rhizobia species The salt tolerant and heavy metals resistance patterns found among the indigenous rhizobial strains associated to G. microcephala and A. uniflorum are reflecting the environmental stresses pressure and make the strains good candidates for plant successful inoculation in arid areas. Our findings indicate that both fast- and slow-growing rhizobia nodulated G. cinerea growing in arid and semiarid regions of northeast Algeria. These rhizobia are poly-extremophiles adapted to diverse environmental stresses and linked to Beta and Gammaprotéobacteria.