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

Abstract

A b s t r a c tFifty 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). 3 332 Guerrouj et al., 2013;Gnat et al., 2014). Surprisingly, Muresu et al. (2008) reported that strains isolated from Hippocrepis unisiliquosa are identified as members of the genus Bacillus or as uncultured bacteria.At Tunisia, Astragalus and Hippocrepis nitrogenfixing symbiotic associations are poorly documented (Zakhia et al., 2004;Mantelin et al., 2006). Previous research's reported that rhizobia associated to Astragalus glombiformis, Astragalus armatus, Astragalus corrugatus and Astragalus algerianus were assigned to the genus of Rhizobium and Phyllobacterium, and only one isolate from nodules Hippocrepis areolata was affiliated to the genus Sinorhizobium (Zakhia et al., 2004).Considering the potential value of the Astragalus and Hippocrepis species in the arid regions of Tunisia and the little information available about the diversity of their root nodulating bacteria, the present paper aim to determine the taxonomic diversity of 57 bacterial collection isolated from root nodules of A. corrugatus and H. areolata by using polyphasic approach including phenotypic and PCR-RFLP analysis and 16S rRNA gene sequencing. Experimental Materials and MethodsBacterial isolation and growth conditions. Fifty seven isolates and six reference strains (Table I) representing different rhizobial species belonging to Rhizobium, Sinorhizobium and Mesorhizobium were used in this study. Rhizobial bacteria were isolated from naturally occurring root nodules collected in four arid soils of Tunisia (Table I). For rhizobia isolation, healthy nodules dissected from roots were surface sterilized with ethanol (70%) and sodium hypochlorite (2%). Then nodules were separately crushed and the nodule juice was streaked on plates of yeast-mannitol agar (YMA) (Vincent, 1970) and incubated at 28°C for the isolation of the rhizobia. The obtained bacterial colonies were purified by being repeatedly streaked on the same medium. Pure isolates were stored with 25% (wt/vol) glycerol at −80°C.Nodulation test. To assess nodulation, seeds were surface-sterilized in 98% sulphuric ...

The ACC-Deaminase Producing Bacterium Variovorax sp. CT7.15 as a Tool for Improving Calicotome villosa Nodulation and Growth in Arid Regions of Tunisia

“…Concerning Rhizobium, our isolates were closely related to R. sullae, which was known to nodulate Hedysarum coronarium L. [40], and to R. pakistanense, recovered from groundnut nodules in Pakistan [41]. Ensifer strains were related to E. meliloti, a common microsymbiont of most wild legumes in Tunisian arid soils [39,42,43]. Isolates belonging to Phyllobacterium have been previously described to nodulate wild legumes in Tunisia [7,38,43].…”

Phylogenomic Review of Root Nitrogen-Fixing Symbiont Population Nodulating Northwestern African Wild Legumes

“…A polyphasic approach analysis indicated that bacterial strains isolated from the pasture legume Biserrula pelecinus growing in Morocco belong to the genus Mesorhizobium. At Tunisia, Mahdhi et al [47] showed that five strains isolated from Astragalus corrugatus were phylogenetically related to M. temperatum and to Mesorhizobium sp. From the tribe Galegeae (subtribe Coluteinae), Ourarhi et al [48] reported that Colutea arborescens is nodulated by diverse rhizobia in Eastern Morocco, among them, the genus Mesorhizobium.…”

“…E. meliloti and E. numidicus strains were isolated from the Genisteae tribe such as Argyrolobium uniflorum, Retama raetam, and Genista saharae [56][57][58]69]. For Galegae tribe, Mahdhi et al [47] reported that rhizobial symbionts of Astragalus corrugatus are closely related to E. meliloti under Tunisian soils. From the Hedysareae tribe, Mahdhi et al [35] reported that strains isolated from Hedysarum spinosissimum root nodules are closely related to E. meliloti in the infra-arid zone of Tunisia.…”

“…Rejili et al [51] reported that Lotus creticus is also nodulated by N. huautlense in the arid areas of Tunisia. For Galegae tribe, Mahdhi et al [47] reported that rhizobial symbionts of Astragalus corrugatus are closely related to N. galegae under Tunisian soils.…”

“…From the Loteae tribe, E. meliloti and E. numidicus were isolated, at Tunisia, from Lotus creticus[49][50][51]69] and Rhizobium sp. from Hippocrepis areolata[47]. From the Acacieae tribe, genetic characterization of rhizobial bacteria-nodulating Acacia tortilis subsp.…”