Résumé:
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.
