Systematic and Applied Microbiology 42 (2019) 440–447 Contents lists available at ScienceDirect Systematic and Applied Microbiology jou rnal homepage: http://www.elsevier.com/locate/syapm Astragalus algarbiensis is nodulated by the genistearum symbiovar of Bradyrhizobium spp. in Morocco a a a a Soufiane Alami , Hanane Lamin , Omar Bouhnik , Salma El Faik , a a b Abdelkarim Filali-Maltouf , Hanaa Abdelmoumen , Eulogio J. Bedmar , a,∗ Mustapha Missbah El Idrissi a Center for Biotechnology, Biodiversity and Environment, Faculty of Sciences, Mohammed V University, Rabat, Morocco b Department of Soil Microbiology and Symbiotic Systems, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, E-419, 18080 Granada, Spain a r a t i b s c l e i n f o t r a c t Article history: Astragalus algarbiensis is a wild herbaceous legume growing in Maamora, the most important cork oak Received 18 February 2019 forest in northern Africa. It is a plant of great importance as fodder in silvopastoral systems, and in Received in revised form 7 March 2019 the restoration of poor and degraded soils. The purpose of this study was to describe the biodiversity of Accepted 15 March 2019 rhizobia nodulating this plant and determine their identity. Out of 80 bacterial isolates, 56 strains isolated from root nodules of A. algarbiensis were characterized. ERIC-PCR fingerprinting grouped the strains in two Keywords: main clusters containing 29 and 27 isolates, respectively, and the amplified ribosomal DNA restriction Astragalus algarbiensis analysis (ARDRA) generated two different ribotypes. Based on both the ERIC-PCR and ARDRA results, Biodiversity Bradyrhizobium representative strains As21 and As36 were selected for further genetic studies. The nearly complete 16S rRNA gene sequences of As21 and As36 showed that they were closely related to Bradyrhizobium cytisi housekeeping genes T Symbiotic genes CTAW11 with similarity values of 99.84% and 99.77%, respectively. Concatenation of atpD, recA, gyrB Symbiovars and dnaK housekeeping gene sequences indicated that strains As21 and As36 had a 95.22% similarity but T they showed values of 95.80% and 94.97% with B. cytisi CTAW11 , respectively. The sequencing of the symbiotic nodC gene of the two strains revealed 97.20% and 97.76% identities, respectively, with that of T B. cytisi CTAW11 isolated from Cytisus villosus growing in the Moroccan Rif Mountains. Furthermore, the phylogenic analysis showed that the strains isolated from A. algarbiensis clustered with B. cytisi and B. rifense within the bradyrhizobia genistearum symbiovar and may constitute two novel genospecies. © 2019 Elsevier GmbH. All rights reserved. Introduction quality and fertility, as well as protection against desertification, is an ecologically sustainable approach that respects the environment With a total area of 132,000 hectares (ha), including 66,000 ha [35]. Legumes are known to be pioneer species and can rapidly of cork oak, the Maamora is the largest cork oak forest in the restore degraded ecosystems [27] through a symbiosis they elicit south Mediterranean region, playing important economic, social with nitrogen (N2)-fixing bacteria called rhizobia [11,25,42,53,64]. and environmental roles [5,17]. This forest, however, is under high The Fabaceae is the third family of the flowering plants with edaphoclimatic and anthropic pressures, since it is submitted to almost 19,000 referenced species and 750 genera [2]. However, different constraints such as overgrazing, climatic changes and only a few leguminous plant species have been studied for nodu- mineral element poverty of its soils [18]. These factors provoke ero- lation, most of which are those used for animal and human sion and desertification and, therefore, degradation and regression consumption [30]. Wild legumes, such as astragals, are charac- of the forest [37,49]. terized by their excellent adaptation to edaphoclimatic conditions The vegetation cover of the Maamora area is known for its great (e.g. drought, salinity, degraded soils) and their multiple uses (e.g. diversity, and Fabaceae plants occupy second place in importance the fight against erosion, animal feed, pastoral, phytoremediation) after the Poaceae plant family [1]. The use of legumes to improve soil [22,23,65]. Astragalus is the largest genus in the Fabaceae with approxi- mately 2900 species [67], and it is widely distributed in Africa, ∗ South America and the Northern Hemisphere [43]. Many species Corresponding author. E-mail address: [email protected] (M. Missbah El Idrissi). of this genus are used in traditional and modern medicine to cure https://doi.org/10.1016/j.syapm.2019.03.004 0723-2020/© 2019 Elsevier GmbH. All rights reserved. S. Alami et al. / Systematic and Applied Microbiology 42 (2019) 440–447 441 several diseases such as diabetic nephropathy and hemorrhoids, rod. The resulting suspension was streaked in Petri dishes contain- and some astragalosides have an anti-aging effect related to the ing yeast extract-mannitol (YEM) medium [58] supplemented with ◦ improvement of brain function and immunomodulatory effects. 0.0025% (w/v) Congo red. After incubation of the plates at 28 C They also constitute an important source of pharmacologically for 10 d, colony forming units representing all the colony types active molecules with hepatoprotective, immunostimulatory and that could be distinguished by microscopic observation of living antiviral effects [55]. cells were chosen. After identification, all the isolates used in the In the Mediterranean area, nearly 500 species of Astragalus have study were routinely grown in tryptone yeast-extract (TY) medium been reported [45]; however, only three spontaneous species are to prevent the overproduction of exopolysaccharide produced on recorded in the Maamora forest, A. algarbiensis Coss. A. lusitanicus YEM medium [4]. Lam (R) and A. solandri Lowe (N) [1]. A. algarbiensis (syn. Tragacantha Before further analysis, the symbiotic property of each isolate algarbiensis) is an annual wild herb, endemic to Morocco, Algeria was checked by the possession of the nodC nodulation gene. The and the Iberian Peninsula. According to the European Committee nodC gene was amplified by PCR, as mentioned subsequently, and for the Conservation of Nature and Natural Resources [52], A. algar- isolates that failed amplification were excluded. The final number biensis is in the course of extinction in Portugal and Spain, but it of A. algarbiensis isolates was thus reduced to 56. is widely distributed in the sandy substrates of the coastal regions of Morocco [5,44,54] where it is a multipurpose plant used as fod- Phenotypic tests der for livestock and to prevent soil erosion and desertification. In addition, A. algarbiensis can enrich the soil with nitrogen due to its Tests were performed in liquid or on solid media inoculated with ability to fix atmospheric N2 in symbiosis with local rhizobia. an exponentially growing liquid culture. The tolerance of the iso- Rhizobia are aerobic soil bacteria that form N2-fixing sym- lates to high temperatures was tested on TY medium at 28, 35, bioses with leguminous plants by eliciting nodules on the ◦ 40, 43 and 45 C. The ability of isolates to grow on acidic or basic roots, and sometimes on the stems, of their host plants. To media was determined on solid YEM medium whose pH had been date, rhizobia have been classified into the genera Rhizobium, adjusted and buffered to 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5 or 9, as described Ensifer (formerly Sinorhizobium), Allorhizobium, Shinella, Pararhizo- by Zerhari et al. [68]. The salt tolerance of the isolates was tested bium, Neorhizobium, Mesorhizobium, Phyllobacterium, Aminobacter, on TY medium supplemented with 1.7, 85, 171, 342, 513, 684 and Bradyrhizobium, Blastobacter, Photorhizobium, Devosia, Azorhizo- 855 mM NaCl. bium, Methylobacterium, Microvirga and Ochrobacterium within the Alphaproteobacteria, together with Paraburkholderia, Burkholderia and Cupriavidus (formerly Ralstonia) within the Betaproteobacte- DNA extraction and ERIC-PCR fingerprinting ria [56]. Many studies have shown that legumes have an intrinsic capacity to accommodate both symbiotic and non-symbiotic endo- For DNA extraction and PCR amplifications, genomic DNA was phytic bacteria within root nodules [15,52,66]. isolated from bacterial cells as previously described [26]. The Nitrogen fixing ability has been recorded in approximately 90 quantity of DNA was determined by using a NanoDrop spectropho- species of Astragalus [2], which are known to be nodulated by differ- tometer (NanoDrop ND2000/2000c, Thermo Fisher Scientific, USA). ent species of the genera Rhizobium [11,12,19,32,41,62,70], Ensifer Enterobacterial repetitive intergenic consensus (ERIC)-PCR was [11,19,41,64,69] and Mesorhizobium [11,19,20,21,41,60,61,64,70]. performed as indicated earlier [25], using primers ERIC1R and ERIC2 In addition, members of the slow growing genus Bradyrhizobium [57] to reduce the number of strains and avoid any duplicates have been shown to establish N2-fixing symbiosis with A. henryi, A. or clonality. Comparative analyses of the electrophoretic ERIC- melilotoides and A. sinicus [10,70]. PCR patterns were performed with GelCompar II software (version The analysis of the biodiversity of rhizobial populations associ- 2.5 Applied Maths, Belgium) by using the unweighted pair group ated with Astragalus will permit the selection of efficient inocula method with arithmetic averages (UPGMA). that could be utilized to improve the growth of this legume in different