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,

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 road, Fez. Bean 10 S7 , 41 kilometres north-west of Fez in the Atlas Mountains. Bean 8 S8 Trapping of rhizobia from ’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 : , 23 km from Fez. Bean + Chickpea 7 S12 Moulay Driss Zerhoun, 90 km from Fez, 25 km from 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 7 days at 28°C, single colonies were selected and restreaked on YMA for purity (Jordan, 1984). Pure The colony morphology of the isolates was examined on yeast cultures were preserved in 20% glycerol at -80°C until further use extract mannitol (YEM) agar plate. After an incubation of 3 to 7 (Elbanna et al., 2009; El-Akhal et al., 2009). days at 28°C, individual colonies were characterized based on their size, color, shape, mucosity, transparency, borders, elevation, and Gram stain reaction (Vincent, 1970). Authentication of isolates

Authentification experiment was conducted in Gibson tubes. All Bromothymol blue test isolates were tested for nodulation ability on the host legume. Experiments were carried out in a growth chamber at 28°C. Seeds All the isolates were tested on YEM agar containing 0.025% were surface sterilized by rinsing in ethanol 95% (v/v), soaking for 4 bromothymol blue for 3 to 10 days for acid or alkaline reaction min in HgCl2, followed by three washings in sterile distilled water (Vincent, 1970). A color change after 72 h at 28°C to yellow was and were scarified with H2SO4 at 95%. Later, seeds were recorded as acid production. germinated in sterilized dishes containing sterile damp filter paper and sterile distilled water was added at intervals to keep the filter paper and germinating seeds wet. Seeds were incubated at 28°C Salt tolerance test for 2 to 3 days until radicals were 2 to 3 cm long and root hairs appeared. The seedlings were placed aseptically in Gibson tubes Rhizobia were examined for their tolerance to salt on yeast extract supplemented with a nitrogen free plant nutrient solution (Gibson, mannitol agar (YMA) supplemented with 0.5, 1, 1.5, and 2% (w/v) 1980). Each tube was inoculated with a rhizobial suspension from NaCl (Ben Romdhane et al., 2006). Berrada et al. 5317

Figure 2. Electrophoresis of PCR products obtained with the universal primers S-D-Bact-0008-a-S-20 and S-D- Bact- 1495-a-A-20.

pH tolerance test following the Ivanova et al. (2000); with slight modifications. The rhizobial cultures were resuspended in 267 ml of Tris-EDTA (TE) The ability of the Rhizobium isolates to grow on media at several buffer and 30 µl lysozyme (30 mg/ml freshly prepared), and were pH values was tested by streaking cultures on the YMA plates, incubated at room temperature for 30 min at 37°C. 3 µl of where the pH values were adjusted to 4.8, 5.8, 6.8, and 8.8 with proteinase K (20 mg/ml) was added, mixed well and incubated at either NaOH or HCl (Küçük et al., 2006). 37°C for 30 min. 40 µl of 10% SDS was added, mixed and incubated at 37°C for 30 min. After incubation, the cell lysates were added with 100 µl of 5 M NaCl; 80 µl of cetyltrimethylammonium Temperature tolerance bromide (CTAB) (CTAB 10%, NaCl 0.7 M) and incubated at 65°C for 10 min. The cell lysate was deproteinized with an equal volume Temperature tolerance was tested by incubating the inoculated of phenol/chloroform/isoamyl alcohol, and was centrifuged at 12000 plates at 4, 20, 27, 37 and 55°C (Hung et al., 2005). rpm for 20 min at 4°C. The aqueous layer was transferred carefully to a fresh tube, the volume was noted; added with an equal volume of Intrinsic antibiotic resistance chloroform/isoamyl alcohol and centrifuged at 12000 rpm for 20 min at 4°C. The aqueous phase was transferred to a fresh tube; added All isolates were treated with selected antibiotics to determine their with 2.5 v/v ethanol and incubated at -20°C for overnight. The intrinsic antibiotics resistance pattern. The antibiotics used were precipitated DNA was pelletized by centrifuge at 12000 rpm for 30 (µg/ml): chloramphenicol (10, 50, 100), streptomycin (10, 50), min at 4°C. After discarding the supernatant, the pellet was vacuum kanamycin (10), ampicillin (100, 200), and tetracycline (10, 50). dried for 30 min and resuspended in 30 µl of TE buffer (Rajasundari Stock solution of the antibiotics was prepared immediately before et al., 2009; Ben Romdhane et al., 2005). use in sterile distilled water with the exception of tetracycline which was dissolved in a 70% ethanol. Appropriate quantities of the antibiotic stock solutions mentioned earlier were added to molten 16S RNA gene amplification YMA, mixed thoroughly and then poured on petri dishes. Isolates were grown in YM broth for 48 h and 10 µl of each isolate were The prokaryotic specific primers used for 16S rRNA gene inoculated on a petri dish and incubated at 28°C for 7 days (Kenenil amplification were S-D-Bact-0008-a-S-20 F (5′ et al., 2010). AGAGTTTGATCCTGGCTCAG 3′) and S-D-Bact- 1495-a-A-20 R

(5’ CTACGGCTACCTTGTTACGA 3’) (Figure 2). PCR amplification

was carried out in a 25 µl reaction volume-containing template DNA Intrinsic resistance to heavy metals (100 ng), Taq buffer (5x), MgCl2 (25 mM), dNTP mixture (25 mM), This test was conducted to assess the ability of the isolates to resist forward primer (25 µM), reverse primer (25 µM), and (5 U/μl) of Taq DNA polymerase. PCR amplification was performed with a Bio-Rad to different types of heavy metals like: AsNa2HPO4 (60, 100, 300), TM CuSO (10, 50, 100), HgCl (20, 25, 100), NiCl , 6H O (20, 40, 60, S1000 Thermal Cycler model. The PCR temperature profile used 4 2 2 2 was 94°C for 4 min followed by 35 cycles consisting of 94°C for 45 80), ZnSO4, 7H2O (10, 20, 40), and K2Cr2O7 (25, 50, 100) (µg/ml). The stock solutions of different metals were sterilized by filtration s, 55°C for 45 s, 72°C for 1 min, with a final extension step at 72°C (Millipore 0.2 µm). The results were evaluated after one week of for 10 min. Reaction efficiency was estimated by horizontal agarose incubation at 28°C (Küçük et al., 2006). gel electrophoresis (1% w/v) and colored in an aqueous solution of ethidium bromide (1 mg/ml) and photographed under ultraviolet (UV) illumination with an apparatus Gel Doc Bio-Rad (Ben Genotypic characterization Romdhane et al., 2006).

The molecular study involved 17 selected bacteria and was carried out at the Laboratory of Microorganisms and Active Biomolecules at 16S rRNA gene sequencing the Faculty of Sciences of Tunis. The 16S rRNA genes were amplified by PCR with bacterial universal primers S-D-Bact-0008-a-S-20 F and S-D-Bact- 1495-a-A- Genomic DNA extraction of rhizobial isolates 20 R. The amplified fragments were purified with a Shrimp Alkaline Phosphatase enzyme. 16S rRNA gene cycle sequencing were The total genomic DNA of rhizobial isolates was isolated by performed using Big-dye terminator kit as recommended by the 5318 Afr. J. Microbiol. Res.

manufacturer, and the nucleotide sequence of the amplified 16S this study can tolerate temperatures ranging between 20 rRNA gene determined by the dideoxy chain termination method of to 37°C, but not above 55°C. The antibiotic resistance of Sanger et al. (1977) by an automatic DNA sequencer. The 16S the isolated strains showed a high level of resistance rDNA sequences were initially analyzed by searching the National Center for Biotechnology Information (NCBI) database using the against streptomycin, ampicillin, tetracyclin, kanamycin, BLAST N program (Altschul et al., 1997). and chloramphenicol. All of the strains of Rhizobium species were tested for their tolerance to six heavy metals. The highest resistance was recorded for RESULTS arsenate, copper, zinc, nickel, and mercury, while a moderate resistance for Chromium was observed. Isolation and morphological characteristics Different physiological characteristics of the 110 rhizobial isolates are summarized in Tables 2 and 3 and A total of 110 strains were isolated from root nodules of Figure 3. Values indicate the number of tolerant isolates. different legumes collected from different sites in the region of Fez. All isolates were subjected to Gram staining and microscopic observation showed that the Genotypic characterization studied isolates were all Gram negative. Approximately, the majority of rhizobial isolates had the same colony The rRNA gene sequences revealed the general morphology and growth rate on YMA medium. A high evolution history of bacteria, although lateral transfer of production of mucus was verified in 81% of the studied rRNA genes may have happened among different isolates. They formed transparent to creamy colonies bacteria. Based on the results of the symbiotic with 2 to 4 mm in diameter after 1 to 3 days incubation on effectiveness tests conducted in a Gibson tube petri YMA plates. experiment and phenotypic characterization, the 17 most representative isolates: S4F(3)T, S5F(I), S7F(3), S8TL2, S8TT2tr, S9F(II), S10F(I), S10F(II), S11F1(I), S11F(III), Authentication of isolates S13F3(I), S13PC(6), S14F1(I), S14F7(II), S14F10(II), S14F10(III), and S15a2 were chosen for further genotypic Testing isolates for their ability to nodulate roots of the characterization. The 16S rDNA were amplified and a target legume and for their symbiotic effectiveness under single band of about 1500 bp for all the strains was sterile conditions is of prime importance (Subba Rao, obtained (Figure 2), which corresponded to the expected 1977). In the Gibson tubes experiment, conducted in the size of the 16S rRNA gene (Amrani et al., 2010). present study under growth chamber conditions, the 110 rhizobial isolates from root nodule’s plants were tested for their ability to nodulate roots (authentication as rhizobia). Sequence analysis The effective nodulation observed with all rhizobial isolates clearly indicated that all 110 isolates were able to To examine the taxonomic status of the isolates in more induce nodulation of plant roots. It was noted that the detail, 17 strains representing a diversity of isolates in the nodules were pink, indicating leghemoglobin content, and 16S rDNA genotype groups, were selected for sequence the leaves of the nodulated plants were dark-green, while analysis. The result of the sequence analysis of 16S uninoculated unfertilized control plants were yellow. rDNA in Table 4 indicated that 6 strains (S11F1(I); S5F(I); S14F1(I); S8TT2tr; FS10(II) and S14F10(II)) had 100% sequence identity with Rhizobium sp., accession Phenotypic characterization numbers EF549399.1, EF549389.1, EU529842.1, JN944178.1, AB529851.1, and JF900025.1. 5 strains Differences between strains were verified using some (S13F3(I); S14F7(II); S14F10(III); S9F(II); S15a(2)) had 99% morphological parameters. Most rhizobial strains were sequence identity with Rhizobium sp., accession fast-growing rhizobia (79%), 16% were intermediate numbers HM179132.1, FR870233.1, AB529850.1, growing and 5% were slow growing. The neutral to HQ891956.1, JF900028.1. while 1 strain (S8TL2) had alkaline pH were the most tolerated. Indeed, 79% of the 98% similarity with Rhizobium sp., accession number isolates were able to tolerate a pH 8.8. At neutral pH, all EU741078.1. However, 16S rDNA from strains S10F(I) isolates showed optimal development (96%). Tolerance and S11F(III) had successively 100 and 99% sequence to acidity was variable depending on the tested isolate: identity with Rhizobium leguminosarum, accession we noticed a decrease in tolerance from neutrality to numbers HQ218435.1 and JN180926.1. 2 strains (S7F(3) acidity. However, 88% of the isolates could grow at pH and S13PC(6)) were classified as Agrobacterium species, 5.8 and 69% of the isolates were able to tolerate pH 4.8. accession numbers EF427851.1 and EF427855.1 with The growth responses of total strains to NaCI at 0.5, 1, similarity level between 99 to 100%, (Table 4). Also, our 1.5, and 2% are shown in Table 2. 31% of the strains analyses showed that the 16S rDNA from strain: S4F(3)T, were tolerant to 2% NaCI. It was found that the isolates of was identified as Rhizobium radiobacter (formally Berrada et al. 5319

Table 2. Phenotypic characteristics of strains under environmental stresses.

Site Characteristic S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 S15 S16 n=8 n=11 n=1 n=6 n=4 n=10 n=8 n=12 n=2 n=5 n=7 n=2 n=14 n=16 n=2 n=2 Fast growth 8 10 1 5 3 8 8 10 2 4 7 1 10 8 2 0 Intermediate growth 0 1 0 1 1 2 0 2 0 1 0 0 3 5 0 1 Slow growth 0 0 0 0 0 0 0 0 0 0 0 1 1 3 0 1

Growth pH 4.8 6 3 1 3 1 8 5 9 2 4 5 2 12 12 1 2 5.8 5 6 1 4 2 10 8 11 2 5 7 2 14 16 2 2 6.8 8 9 1 6 4 9 8 12 2 5 7 2 14 15 2 1 8.8 8 7 1 6 3 7 7 9 2 4 6 2 12 12 1 0

Growth temperature 4 8 3 0 2 0 6 6 8 2 4 4 2 3 8 2 2 20 8 11 1 6 4 10 7 8 2 5 7 2 12 13 2 2 30 8 11 1 6 4 10 8 12 2 5 7 2 14 16 2 2 37 8 10 1 6 4 10 8 12 1 4 4 1 4 16 2 2 55 1 2 1 2 3 5 1 2 1 1 1 0 0 2 0 2

Growth salinity (%) 0.5 7 8 0 6 4 10 8 11 2 5 7 2 14 14 2 2 1 5 5 0 4 2 5 4 7 1 3 7 0 2 14 2 1 1.5 4 3 0 3 2 4 2 5 0 1 6 0 1 13 2 0 2 2 2 0 2 2 4 1 3 0 1 5 0 1 9 2 0

Table 3. Antibiotic resistance of the isolated strains.

Site Antibiotic (µg/ml) S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 S15 S16 n=8 n=11 n=1 n=6 n=4 n=10 n=8 n=12 n=2 n=5 n=7 n=2 n=14 n=16 n=2 n=2 Amp 100 5 8 0 4 1 5 6 6 1 4 3 1 10 13 2 1 Amp 200 3 6 0 3 0 2 5 5 0 3 2 0 8 11 1 0 Tetr 10 4 8 1 5 2 6 6 9 1 5 4 2 11 12 1 0 Tetr 50 3 6 0 3 1 5 4 8 0 3 2 0 8 6 1 0 Strep 10 8 10 1 6 3 10 7 11 0 4 5 2 10 14 2 0 Strep 50 6 7 0 4 2 9 6 10 0 4 3 1 7 10 1 0 Chlr 10 8 9 1 5 4 8 6 10 1 5 6 1 12 13 0 1 Chlr 50 7 6 1 4 3 7 5 7 0 3 4 0 10 9 0 1 Kan 10 7 10 0 4 3 9 7 11 1 4 7 0 13 10 2 1 Kan 25 6 8 0 2 3 7 5 10 1 2 5 0 12 9 1 0

Agrobacterium tumefaciens), with 100% similarity to rhizobia strains in Fez. Phenotypic and genotypic accession number X67223.1. methods were used to evaluate these natural rhizobia. In this study, a high diversity among rhizobial strains was found. The results of this study corroborated several DISCUSSION studies, which revealed a high heterogeneity in the populations of legume nodulating rhizobia (Ndiaye, 1996; This study provides the first clear characterization of Khbaya et al., 1998; McInroy et al., 1999; Mohamed et 5320 Afr. J. Microbiol. Res.

(%)

esistance esistance R

Heavy metals (µg/ml)

Figure 3. Resistance of strains against heavy metals.

Table 4. Sequence analysis of 16S rDNA from selected Moroccain Rhizobium isolates.

Strain Host plant Identification Similarity (%) Accession number

S11F1(I) Bean Rhizobium sp. 100 EF549399.1

S5F(I) Bean Rhizobium sp. 100 EF549389.1

S10F(I) Bean R. leguminosarum 100 HQ218435.1

S14F1(I) Bean Rhizobium sp. 100 EU529842.1

S8TL2 Alfalfa Rhizobium sp. 98 EU741078.1

S11F(III) Bean R. leguminosarum 99 JN180926.1

S13F3(I) Bean Rhizobium sp. 99 HM179132.1

S8TT2tr Alfalfa Rhizobium sp. 100 JN944178.1

S7F(3) Bean Agrobacterium sp. 100 EF427851.1

FS10(II) Bean Rhizobium sp. 100 AB529851.1

S14F7(II) Bean Rhizobium sp. 99 FR870233.1

S14F10(III) Bean Rhizobium sp. 99 AB529850.1

S4F(3)T Bean A. tumefaciens 100 X67223.1

S9F(II) Bean Rhizobium sp. 99 HQ891956.1

S13PC(6) Chickpea Agrobacterium sp. 99 EF427855.1

S15a(2) Acacia Rhizobium sp. 99 JF900028.1

S14F10(II) Bean Rhizobium sp. 100 JF900025.1

al., 2000; Lafay and Burdon 2001; Ben Romdhane et al., (Küçük et al., 2006). A symbiotic association between 2006; Shamseldin et al., 2009). rhizobia and legumes may be accidental or a simple Most of the isolates in this study (81%) were able to physical association without any mutual benefit to both produce mucous, which might protect them from organisms (Wilkinson, 2001). The results obtained from desiccation and help them to withstand temperature, nodulation test showed that all isolates nodulated their salinity and pH fluctuations. The isolates which exhibited host plant. Salinity is one of the major factors restricting a wide adaptation to these stresses could be able to symbiotic nitrogen fixation. Rhizobia may grow at higher circumvent limiting factors and may become suitable salt levels than their host plant species (Singleton et al., candidates for commercial applications. 1984). Salt-stress leads to changes in In this study, most rhizobial strains were fast-growing exopolysaccharides of rhizobia which helps these rhizobia (79%); which is consistent with previous studies bacteria adapt to stress condition (Mei-Hua et al., 2005). Berrada et al. 5321

In the present study, many isolates were able to tolerate tolerance of strains to antibiotics is not correlated with salt concentrations above 2% (31% of isolates). Also, we their growth rate, but could be related to the bacterial found that fast growing isolates were generally more species. tolerant to high NaCl concentrations than slow growing All strains of Rhizobium were tested for their tolerance isolates. These results are concordant with Küçük et al. to heavy metals. The highest resistance was recorded for (2006). Hence, these isolates may be the suitable arsenate, copper, zinc, nickel and mercury, this result is candidates for use in saline soils, which are frequently consistent with literature showing that Rhizobium is observed in some irrigated areas in Morocco where the resistant to high concentrations of arsenate, zinc, copper, phenomena of salinity is observed in almost 40.000 ha and even mercury (Carrasco et al., 2005). In genetic (Bensouda, 1995). studies, heavy metal resistance traits should be Most of the isolates in this study could tolerate extremely valuable as positive selection markers. The temperatures ranging between 20 and 37°C with high levels of Zn and Cu (Hungria et al., 2000; Zerhari et optimum growth at 30°C (100%), but some of them were al., 2000) suggest that these metals could be used as also able to grow at 4°C (55%) and at 55°C (22%). The selective agents for some Rhizobium strains. results of this test are in concordance with previous Characteristics relating to origin are probably associated studies (Graham, 1992; Zahran, 1994) which showed that with adaptations to specific environmental pressures. Rhizobium bacteria are mesophiles, and can grow at In general, the phenotypic study showed large temperatures between 10 and 37°C with an optimum physiological and biochemical biodiversity. Indeed, the temperature for growth of most isolates at 28°C. studied strains showed a variable resistance against However, since many other factors influence the stress factors, namely, temperature, pH, salinity, competitiveness and efficiency of strains, in vitro resistance to antibiotics and heavy metals, which allowed selection of temperature tolerant root nodule bacteria is the selection of good candidates for genetic studies. not considered as a promising approach for field Several investigators have studied the genetic diversity of applications (Hungria and Vargas, 2000). The survey of Rhizobium isolated from several countries around the such characteristics can be useful for future improvement world (Amanuel et al., 2000; Mutch et al., 2003; Mutch of inoculants by genetic engineering of strains with high and Young, 2004; Moschetti et al., 2005; Depret et al., efficiency for nitrogen fixation, but not sensitivity to 2004; Vessey and Chemining'wa, 2006; Tian et al., 2007; temperature changes. Shamseldin et al., 2009). Although, a few rhizobia grow well at pH value of less In the studies reported here, the genetic biodiversity of than 5 (Graham et al., 1994), some strains of Rhizobium 17 represented strains were examined: S4F(3)T, S5F(I), tropici, Mesorhizobium loti, Bradyrhizobium species and S7F(3), S8TL2, S8TT2tr, S9F(II), S10F(I), S10F(II), S11F1(I), Sinorhizobium meliloti are very acid-sensitive (Brockwell S11F(III), S13F3(I), S13PC(6), S14F1(I), S14F7(II), S14F10(II), et al., 1995). The optimum pH for the growth of root S14F10(III), and S15a2 using PCR techniques with the nodule bacteria usually falls between 6 and 7 (Jordan, primers: S-D-Bact-0008-a-S-20 F and S-D-Bact- 1495-a- 1984). The rhizobial isolates from tree legumes (Acacia A-20 R. farnesiana, Dalbergia sissoo and Sesbania formosa) The genetic study carried out is a quick method that grew at pH12 (Surange et al., 1997). The data of this gives a better idea of the diversity of these strains. The study showed that the isolates studied are globally 16S rDNA was sequenced to determine the taxonomic tolerant of alkalinity and neutrality. 24 out of 110 isolates position of these strains, and the results revealed that were acid sensitive, but were able to grow at an initial pH there was a great genetic diversity among the 17 of 8.8. Similarly, 16 of the studied isolates were sensitive rhizobial strains studied. Indeed, sequence analysis of to alkaline pH, but were able to grow at an initial pH of 16S rDNA and subsequent BlastN analyses indicated 4.8. The isolates which can survive on a wide pH range that 12 strains had 98 to 100% similarity with Rhizobium are candidates for further strain improvement to highly sp., 2 strains were classified as R. leguminosarum; also, acidic or alkaline conditions. Extremes of pH can be a the results of this study showed that 2 strains had high major factor limiting microorganisms in soil (Brockwell et rDNA identity with Agrobacterium sp., and 1 strain were al., 1991). similar to R. radiobacter (formally A. tumefaciens), but Regarding the intrinsic resistance to antibiotics, could not induce tumour formation. In fact, Agrobacterium different authors have reported effect of antibiotics on sp. is genetically related to Rhizobium genomic species Rhizobium bacteria (Rodriguez et al., 2000; Zerhari et al., and several authors have reported the isolation of 2000; Hungria et al., 2001; Küçük et al., 2008). Also, it Agrobacterium-like bacteria from nodules of different has been reported that fast-growing strains are more legume hosts, including common beans from Moroccan sensitive to antibiotics than slow-growing rhizobia and Egyptian soils (Shamseldin et al., 2005), and other (Maatallah et al., 2002). Conversely, in this study, legume nodules from Pakistan (Hameed et al., 2004). rhizobial strains which are a mixture of fast, intermediate Also, several Rhizobium strains lacking their pathogenic and slow growing showed a wide range of behaviour with gene were isolated from root nodules of tropical legumes regard to antibiotics. These results may indicate that the in Africa (De Lajudie et al., 1999; Khbaya et al., 1998) 5322 Afr. J. Microbiol. Res.

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