440 Chiang Mai J. Sci. 2016; 43(3) Chiang Mai J. Sci. 2016; 43(3) : 440-451 http://epg.science.cmu.ac.th/ejournal/ Contributed Paper Diversity of Bacteria Nodulating Medicago arborea in the Northeast Area of Morocco Kamal Guerrouj* [a], Mustapha Bouterfas [b], Hanaa Abdelmoumen [c] and Mustapha Missbah El Idrissi [c] [a] Institut Sup rieur des Professions Infirmi res et des Techniques de Sant Al Hoceima, Morocco - 32000 [b] Laboratoire des sciences de l’eau, de l’environnement et de l’ cologie, Facult des Sciences, Universit Mohamed Premier, Oujda, Morocco. [c] Laboratoire d’am lioration de la productivit des sols et environnement, Ecole Normale Sup rieure, Universit Mohamed V-Agdal, Av. Mohamed Belhassan El Ouazzani-Takaddoum, Rabat, Morocco. *Author for correspondence; e-mail: [email protected] Received: 28 March 2014 Accepted: 16 June 2015 ABSTRACT A Polyphasic characterization of 30 bacteria isolated from root nodules of Medicago arborea (Medic tree) plants growing in 3 arid soils of the arid northeast region of Morocco was performed. The phenotypic, symbiotic, and cultural characteristics analyzed allowed the description of a broad physiological diversity among the isolates. The phenogram obtained showed 3 main clusters at a similarity coefficient of 78%. The results obtained suggest that the phenotype of these rhizobia might have evolved for adaptation to the local conditions. The genetic characterization which consisted in a rep PCR using ERIC (Enterobacterial Repetitive Intergenic Consensus) primers showed a great diversity and 3 groups and 8 clusters were delimited at a similarity level of 80%. However, the 16S rDNA sequences showed a perfect identity of all the isolates with Sinorhizobium meliloti published sequences. The results showed a relationship between rep-PCR fingerprinting and phenotypic techniques, which are complementary in diversity investigation and allowed revelation of relationships between M. arborea close-related bacterial isolates originated from different geographic location in the northeast area of Morocco. Keywords: Medicago arborea, rhizobium, 16S rDNA, bacterial diversity, ERIC-PCR 1. INTRODUCTION Nitrogen constitutes almost 80% of the in all ecosystems as either free-living organisms atmosphere, but is metabolically inaccessible or in symbiotic association with a number of to plants due to the exceptional stability different plants. These N2-fixing prokaryotes of the triple covalent bond (NN). The ability can be anaerobic, facultative aerobic, aerobic, to catalyze enzymatic reduction of N2 to photosynthetic, or nonphotosynthetic. NH3 is limited to a variety of prokaryotes All carry out N2 reduction by an enzymatic defined as nitrogen-fixing or diazotrophic complex termed nitrogenase. microorganisms, which are widely distributed Rhizobia are soil bacteria capable of Chiang Mai J. Sci. 2016; 43(3) 441 eliciting nodules on leguminous plant El Aioun and Bouarg and one from Harraza roots and/or stems in which the bacteria (Oujda region). fix nitrogen. Rhizobial genera include Chemical analysis are focused on pH, Agrobacterium, Allorhizobium, Aminobacter, conductivity, soils organic matter cotenants, Azorhizobium, Bradyrhizobium, Cupriavidus, Nitrogen and Carbon proportions. Mesorhizobium, Methylobacterium, Microvirga, Samples analysis was performed in soil Ochrobactrum, Phyllobacterium, Rhizobium, tests laboratory, INRA, Oujda, and in the Neorhizobium, Pararhizobium, Shinella, Ciceribacter “Centre Oriental des Sciences et Techniques de l’Eau” and Ensifer (syn. Sinorhizobium) [1]. (COSTE), Faculty of Sciences, Mohammed Medicago arborea (Tree Medic) is a perennial Premier University, Oujda, Morocco. woody leguminous shrub native to the Mediterranean region where it is currently 2.2 Bacterial Strains found growing in rocky dry calcareous soils Bacteria were isolated from the root from the Canary Islands and Balearic Islands nodules of Medicago arborea plants grown in along southern Europe to Asia Minor [2]. Oujda, El Aioun and Bouarg soils from the M. arborea is very drought tolerant (<250 mm northeast area of Morocco. From young annual rainfall) and will also grow well in sandy plants of each soil, 10 nodules were sampled, soils. Therefore it has the appeal of being able washed under running water, then encaged in to provide highly nutritious fodder for its a fine-mesh steel holder and surface sterilized wealth content on digestible proteins, while by immersion in 5% Sodium hypochlorite at the same time protecting fragile soils [3]. for 3 min: and finally washed seven times The northeast Moroccan area is known with sterile distilled water. Each nodule was by its arid climate with huge fields entirely crushed on plates containing yeast extract affected by erosion and soil degradation. mannitol agar (YMA) with 0.0025% (w/v) Harsh edapho-climatic conditions and Congo red [4]. After incubation for 20 days entropic factors contribute on desertification’s at 28°C single colonies were selected and installation and proliferation. transferred several times on the YMA plates The characterization of nitrogen-fixing to ascertain purity. A total of 30 pure isolates soil bacteria in symbiosis with Medicago arborea were conserved on YEM slants at 4°C for is envisaged to foster knowledge in the routine analysis and in glycerol at -80°C in ecological behavior of semi-arid soil species El Zaidin Experimental Station, CSIC, and will provide the potential to promote Granada, Spain. plant growth in the harsh conditions prevailing in Mediterranean region. The aim of this work 2.3 Plant Nodulation Tests was to analyze the phenotypic and genetic Seeds of M. arborea were surface sterilized diversity of bacterial isolates from root with 96% ethanol for 30 s followed by nodules of M. arborea grown in 3 different immersion in 15% (v/v) H2O2 for 8 min, soils of the northeast region of Morocco. thoroughly rinsed with sterile distilled water, and allowed to germinate at 30°C in 2. MATERIALS AND METHODS the dark. Isolates were tested for their ability 2.1 Soil Analysis to reinfect M. arborea seedlings. Inoculation Three soils were selected for this study, and seed treatment were performed using all from the arid area of northeast Morocco; the method of Vincent [4]. The plants were two from agricultural technique centre of cultured in a growth chamber under a constant 442 Chiang Mai J. Sci. 2016; 43(3) ° temperature of 23 C and a photoperiod of (1000), MgCl2 (1000), MgSO4 (1000), NiCl2 × × 16 h (light) : 8 h (dark) and were watered 6H2O (250), BaCl2 2H2O (1000), and with nitrogen-free nutrient solution. Indirect CoSO4 (150). Antibiotic resistance of the effectiveness of the nodules for nitrogen isolates was tested on solid TY medium fixation was estimated by visual assay of red containing the following filter-sterilized leghemoglobin presence in cross-sections antibiotics (μg/ml): ampicillin (20), and by the dark green intensity of the leaves carbanicilline (20), chloramphenicol (30), in comparison with uninoculated control gentamycin (10), geneticin (20), neomycin (20), plants. rifampicin (20), spectinomycin (20), nalidixic acid (20), and tetracyclin (50). 2.4 Phenotypic Characterization Hydrolysis of urea by the isolates was A total of 69 phenotypic assays were investigated on solid YEM medium amended tested on the 30 strains isolated from root with 2% (wDv) urea and 0,0012% (wDv) nodules of M. arborea. Inoculations were made phenol red as previously described [8]. with exponentially growing liquid cultures. Gelatinase activity, reduction of nitrate and Tolerance of the isolates to temperature was catalase activity were determined as indicated tested on liquid TY medium [5] at 30°C, by Missbah El Idrissi [9], Lindstrom and 40°C and 45°C, respectively. Ability to grow Lehtomaki [8], and Graham [10], respectively. in acid and basic media was determined For evaluation of oxidase activity, the in YEM medium whose pH had been isolates were spread out over a piece of filter adjusted and buffered to 5.0; 6.0; 7.0; 8.0 paper soaked in a solution containing 1% N, and 9.0, respectively, as described earlier [6]. N-dimethyl-p-phenylendiamin oxalate 98%. For salt tolerance determination they were The appearance of violet colonies indicated grown in 0, 171, 350, 513, 690, 861, and 1000 the presence of oxidase activity. Ability of the mM NaCl. isolates to produce melanin was studied after Utilization of 18 carbohydrates as sole growth on solid TY medium supplemented μ carbon source was investigated on modified with CuSO4 (40 g/ml) and L-tyrosine solid YEM medium as previously reported (600 μg/ml) as previously described [11]. [7]. The monosaccharides glucose, galactose, Computer cluster analysis of the 69 fructose, D-arabinose, ribose, mannose and phenotypic traits tested was carried out for rhamnose, the disaccharides saccharose, the bacterial isolates. The resemblance between lactose, salicine and maltose, the trisaccharides pairs of isolates was calculated using the trehalose and raffinose, the polysaccharides Pearson correlation coefficient and plotted starch and dextrin, the organic acids sodium as a dendrogram with the unweighted acetate and D-gluconic acid, and the pair-group method with arithmetic average polyalcohols mannitol and inositol were (UPGMA) [12]. used. Each compound (1% wDv) was filter- sterilized (0.22 μm) before use. To test 2.5 DNA Extraction and PCR the intrinsic heavy metal resistance, the Amplifications isolates were grown on solid TY medium Bacteria were grown in tryptone-yeast μ × supplemented with