African Journal of Microbiology Research Vol. 6(25), pp. 5314-5324, 5 July, 2012 Available online at http://www.academicjournals.org/AJMR DOI: 10.5897/AJMR11.1505 ISSN 1996-0808 ©2012 Academic Journals Full Length Research Paper Phenotypic and genotypic characterizations of rhizobia isolated from root nodules of multiple legume species native of Fez, Morocco Halima BERRADA1, Imen NOUIOUI2, Mohammed IRAQUI HOUSSAINI1, Naïma EL GHACHTOULI1, Maher GTARI2 and Kawtar FIKRI BENBRAHIM1* 1Laboratory of Microbial Biotechnology, Faculty of Sciences and Technology, Sidi Mohammed Ben Abdellah University, P. O. Box 2202, Fez, Morocco. 2Laboratory of Microorganisms and Active Biomolecules, Faculty of Sciences of Tunis, University campus, P. O. Box 2092. Tunis, Tunisia. Accepted 21 May, 2012 Root-nodulating bacteria were isolated and characterized from grain and forage legumes growing in Fez regions. A total of 110 Rhizobium strains were characterized on the basis of morphological, cultural, and phenotypical properties. Phenotypic characteristics studied included growth rates in various media, colony morphology, tolerances to extremes of temperature, salt and pH and resistance to different concentrations of heavy metals and antibiotics. The isolates were very diverse phenotypically. Eighty seven of isolates were fast growing rhizobia. Thirty four strains tolerated high concentration of salt (2% NaCl). The majority of our isolates tolerated temperatures ranging between 20 and 37°C, but not above 55°C. Also, the isolated strains tolerated extreme pH in their medium from 4.8 to 8.8. The antibiotic resistance of these strains showed a high level of resistance against streptomycin, kanamycin, ampicillin, tetracycline, and chloramphenicol. The highest resistance to heavy metals was recorded for arsenate, copper, zinc, nickel, and mercury. Also, a moderate resistance was found for chromium. Polymerase chain reaction (PCR) method and 16S rRNA gene sequencing were used for the genotypic analysis of 17 Rhizobium strains selected on the basis of phenotypic study. The results showed a high diversity among the strains. Key words: Rhizobium, legumes, nodule, diversity, 16S rRNA. INTRODUCTION Rhizobia have been widely used in agricultural systems Consequently, there has recently been a growing level of for enhancing the ability of legumes to fix atmospheric interest in environmental friendly sustainable agricultural nitrogen (Teaumroong et al., 1998). Nitrogen was known practices and organic farming systems (Rigby et al., to be an essential nutrient for plant growth and 2001; Lee et al., 2007). Increasing and extending the role development. Intensive farming practices that accomplish of biofertilizers such as Rhizobium would decrease the high yields need chemical fertilizers, which are not only need for chemical fertilizers and reduce adverse cost effective, but may also create environmental environmental effects. Development and implementation problems. The extensive use of chemical fertilizers in of sustainable agriculture techniques, such as agriculture is currently under debate due to biofertilization is of major importance in alleviating environmental concern and fear for consumer health. environmental pollution and the deterioration of nature (Ogutcu et al., 2008). Rhizobium symbiosis with legumes species is of special importance, producing 50% of 175 million tonnes of total biological nitrogen fixation, annually *Corresponding author. E-mail: [email protected]. providing nearly half of all N used in agriculture (Hatice et Tel: 00 212 6 61 21 65 98. al., 2008). Berrada et al. 5315 Table 1. Number of isolates obtained from each host plant and corresponding collection sites. Site Description Host plant Colony number S1 Oued (O) Sebou: a few meters upstream of its confluence with O. Fez Bean + Clover 8 S2 O. Sebou: a few meters downstream of its confluence with O. Fez Clover + Lens 11 S3 O. Fez at the exit of the medina Alfalfa 1 S4 Immouzer road, Fez. Bean 6 S5 Fez downtown (across from Sopriam) Bean 4 S6 Sefrou road, Fez. Bean 10 S7 Karia Ba Mohamed, 41 kilometres north-west of Fez in the Atlas Mountains. Bean 8 S8 Trapping of rhizobia from Ain cheggag’s soil Clover 12 S9 Jnanate : near the old city (medina) Bean 2 S10 The medina (Fez old city) Bean 5 S11 Trapping of rhizobia from saline soil : Moulay Yacoub, 23 km from Fez. Bean + Chickpea 7 S12 Moulay Driss Zerhoun, 90 km from Fez, 25 km from Meknes and 5 km from Volubilis. Bean + Chickpea 2 S13 Douyet 10.6 km Western of Fez Bean 14 S14 Annoceur, 80 km to the South of Fez in the middle Atlas. Bean 16 S15 Sidi harazem, 30 km Eastern of Fez Acacia 2 S16 Ain cheggag, 20 km in the South of Fez Clover 2 The taxonomy of bacterial endosymbionts of characteristic patterns when distinguished in agarose leguminous plants has experienced a profound series of gels, providing well separation on strain level (Adiguzel, extensions in the recent past (Young, 2003). Currently, 2006). Recently, wild legumes and their symbionts have there are seven genera of rhizobia containing about 40 drawn the attention of ecologist, because of their species as Alpha-proteobacteria: Allorhizobium, tolerance to extreme environmental conditions, such as Azorhizobium, Bradyrhizobium, Mesorhizobium, severe drought, salinity, and elevated temperatures. In Rhizobium, Sinorhizobium (Wei et al., 2002) and a addition, symbiotic rhizobia of naturally growing legumes species in the genus Methylobacterium (Sy et al., 2001). successfully establish effective symbioses under these New lines that contain nitrogen-fixing legumes symbionts conditions (Zahran, 2001). The objective of this study include Ochrobactrum (Ngom et al., 2004; Trujillo et al., was to isolate and characterize the rhizobial populations 2005), Devosia (Rivas et al., 2003), Blastobacter (Van naturally associated with food and forage legumes Berkum and Eardly, 2002) and Methylobacterium (Jaftha originating from different sites in regions of Fez and by et al., 2002; Jourand et al., 2004) in the alpha- several approaches, including the evaluation of Proteobacteria; Burkholderia (Moulin et al., 2001), phenotypic properties as well as genotypic Cupriavidus (Vandamme and Coenye, 2004; Chen et al., characteristics. 2001) in the beta-Proteobacteria; and some unclassified strains in the gamma-Proteobacteria (Benhizia et al., 2004) were recently described. MATERIALS AND METHODS The design of the diversity of rhizobia is however far from clear, particularly, thinking to the large number of Collection of rhizobia from nodulated legumes leguminous species and their wide geographical The collection of nodulated plants was conducted during the spring distribution (Wei et al., 2002). Otherwise, Morocco has a of years 2008/2009 and 2009/2010 in 16 different sites in the city of great floristic diversity (Elyousfi and M’hirit, 1998) in spite Fez and regions (Table 1 and Figure 1). Each sample constitutes 5 of the increasing pressure on natural resources and hard healthy, green plants collected from 5 random locations in the field. climatic conditions which generate terrestrial ecosystem A clean spade was used to dig approximately 15 cm to either side dysfunctions (Fikri Benbrahim et al., 2004). Since of the plant stalk to a depth of at least 20 cm. The clump of soil and roots was lifted out carefully, placed in clean plastic bags and kept rhizobia are taxonomically very diverse (Wolde-Meskel et in an ice box. Isolation of bacteria was performed the day after al., 2004), efficient strain classification methods are sample collection. needed to identify genotypes displaying, such as, Rhizobium strains were isolated from root nodules of the superior nitrogen-fixation capacity (Sikora et al., 2002). legumes: food (bean, chickpea and lens); forage (alfalfa and Molecular techniques have helped to develop easy and clover); and shrubby (acacia). quick methods to microbial characterization; including works distinguish genera, species and even strains Isolation of rhizobia from nodules (Schneider et al., 1996; Giongo et al., 2008). The polymerase chain reaction (PCR) can create highly To isolate rhizobia from legumes: healthy, unbroken and pink root 5316 Afr. J. Microbiol. Res. Figure 1. Map showing the collection sites of legume nodules (http://www.google.com/mapmaker?ll=33.952474,- 5.177307&spn=0.610581, 1.870422&z=10&lyt=large_map&htll=34.332096,-5.668945). nodules were selected. Rhizobia were isolated from fresh surface an early stationary-phase culture. Uninoculated plants were used sterilized nodules by the standard method (Van Berkum et al., as controls. Three replicates were prepared to each treatment. After 1996). Hence, nodules were immersed in 95% ethanol (v/v) for 10 s a month, the plants were harvested and the number of nodules was and were surface sterilized in HgCl2 for 4 min, and were washed determined. The nitrogen fixation ability of the strains was three times in autoclaved distilled water. Effectiveness of estimated from the pink color of the nodules and the dark green sterilization was monitored to eliminate the possibility of isolating color of leaves compared to the control plants. surface-attached bacteria. Sterilized nodules were crushed with a sterile glass rod in a sterile test tube, using some drops of NaCl (9‰) to make it slurry (Beck et al., 1993). One loop full of the Phenotypic characterization nodule content suspension was streaked on yeast mannitol agar (YMA) plates (Vincent, 1970) containing 0.0025% (w/v) Congo red. Colony morphology After incubation for 3 to