MOSBAH MAHDHI¹, NADIA HOUIDHEG², NEJI MAHMOUDI², ABDELHAKIM MSAADEK², MOKHTAR REJILI² and MOHAMED MARS²

¹Center for Environmental Research and Studies, Jazan University, Jazan, Kingdom of Saudi Arabia
²Research Unit Biodiversity and Valorization of Arid Areas, Bioressources (BVBAA), Faculty of Sciences, Gabès University, Erriadh-Zrig, Gabès, Tunisia

*Corresponding author: M. Mahdhi, Center for Environmental Research and Studies, Jazan University, Jazan, Kingdom of Saudi Arabia; e-mail: mosbahtn@yahoo.fr.

Submitted 13 June 2015, revised 11 October 2015, accepted 11 February 2016
DOI: 10.5604/17331331.1215612

Abstract

Fifty 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)

Key words: 16S rDNA sequencing, arid areas, PCR-RFLP, phenotypic properties, rhizobial bacteria

Introduction

Rhizobia or “legume nodulating bacteria (LNB)” or “root nodule bacteria’ (RNB)” are defined as nitrogen-fixing bacteria that form nodules on legume plants. In the last few years, a large diversity of LNB has been revealed, which has caused deep changes in the taxonomy of these bacteria. Rhizobia currently consist of 98 species belonging to 13 different genera. The predominant symbionts for most legume species in habitats throughout the world are found in the a-class of Proteobacteria: Rhizobium, Azorhizobium, Ensifer (formerly Sinorhizobium), Mesorhizobium, Bradyrhizobium, Methylobacterium (Jaftha et al., 2002; Jourand et al., 2004), Devosia (Rivas et al., 2003), Shinella (Lin et al., 2008), Ochrobactrum (Trujillo et al., 2005; Zurdo-Pineiro et al., 2007), Phyllobacterium (Valverde et al., 2005; Mantelin et al., 2006) and Microvirga (Ardley et al., 2012). Moreover, about eight species within two genera of ß-class of Proteobacteria – Burkholderia and Cupriavidus have been reported (Moulin et al., 2001; Chen et al., 2001; 2006; 2008; Klonowska et al., 2012). In addition, bacteria from γ-class of Proteobacteria have also been reported (Benhizia et al., 2004; Muresu et al., 2008; Mahdhi et al., 2012). On the other hand, many Agrobacterium-like strains have been isolated from root nodules of different legumes species (Gurtler et al., 1991; Liu et al., 2005; Mahdhi et al., 2008), but all of them failed to nodulate their original plant hosts and until now no definitive explanation of the presence of these bacteria inside nodules could be demonstrated.

Legumes belonging to the genera Astragalus and Hippocrepis are distributed in northern Africa, southern Europe and East Asia. Several Astragalus species are used as herbal medicine and Hippocrepis species have a wide range of uses as minor crops including consumption, fodder, forage and land stabilization. Despite the high number of these legume species (3000 species for Astragalus and 20 for Hippocrepis), only few of them have been considered for their nitrogen symbiotic fixation. Previous studies reported that microsymbionts associated to root nodules of some Astragalus species belonged to Sinorhizobium, Rhizobium, Agrobacterium, Bradyrhizobium and Mesorhizobium (Zhang et al., 2000; Gao et al., 2001; 2004; Wei et al., 2003; Zhao et al., 2012; 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 nitrogen-fixing 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 Methods

Bacterial 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.

Table I
New isolates and reference strains used in this study

Isolates	Site of origin	Host plants	Nodulation test	16S rDNA type
ACM1	Menzel Habib	Astragalus corrugatus	–	1*
ACM2	Menzel Habib	Astragalus corrugatus	+(12)	2†
ACM3	Menzel Habib	Astragalus  corrugatus	+(11)	2†
ACM4	Menzel Habib	Astragalus  corrugatus	+(10)	2†
ACN1	Nefta	Astragalus  corrugatus	+(15)	3ᶲ
ACN2	Nefta	Astragalus  corrugatus	+(14)	3ᶲ
ACN3	Nefta	Astragalus  corrugatus	+(11)	3ᶲ
ACN4	Nefta	Astragalus  corrugatus	+(11)	3ᶲ
ACN5	Nefta	Astragalus  corrugatus	+(10)	2†
ACN6	Nefta	Astragalus  corrugatus	+(10)	2†
ACN7	Nefta	Astragalus  corrugatus	+(10)	2†
ACN8	Nefta	Astragalus  corrugatus	+(13)	2†
ACN9	Nefta	Astragalus  corrugatus	+(15)	2†
ACN10	Nefta	Astragalus  corrugatus	–	1*
ACN11	Nefta	Astragalus  corrugatus	+(14)	2†
ACN12	Nefta	Astragalus  corrugatus	+(14)	3ᶲ
ACN13	Nefta	Astragalus  corrugatus	+(13)	3ᶲ
ACN14	Nefta	Astragalus  corrugatus	+(11)	2†
ACN15	Nefta	Astragalus  corrugatus	+(11)	2†
ACN16	Nefta	Astragalus  corrugatus	+(14)	3ᶲ
ACZ1	Zarzis	Astragalus  corrugatus	+(13)	4‡
ACZ2	Zarzis	Astragalus  corrugatus	+(15)	4‡
ACZa1	Zárate	Astragalus  corrugatus	+(15)	4‡
ACZa2	Zárate	Astragalus corrugatus	+(15)	4‡
ACZa3	Zárate	Astragalus  corrugatus	+(12)	4‡
HBM1	Menzel Habib	Hippocrepis areolata	+(12)	3ᶲ
HBM2	Menzel Habib	Hippocrepis areolata	+(13)	3ᶲ
HBM3	Menzel Habib	Hippocrepis areolata	+(14)	3ᶲ
HBM4	Menzel Habib	Hippocrepis areolata	+(11)	3ᶲ
HBM5	Menzel Habib	Hippocrepis areolata	+(10)	3ᶲ
HBM6	Menzel Habib	Hippocrepis areolata	+(10)	3ᶲ
HBM7	Menzel Habib	Hippocrepis areolata	+(11)	3ᶲ
HBM8	Menzel Habib	Hippocrepis areolata	+(12)	3ᶲ
HBM9	Menzel Habib	Hippocrepis areolata	+(13)	3ᶲ
HBN1	Nefta	Hippocrepis areolata	+(14)	3ᶲ
HBN2	Nefta	Hippocrepis areolata	+(12)	3ᶲ
HBN3	Nefta	Hippocrepis areolata	+(11)	3ᶲ
HBN4	Nefta	Hippocrepis areolata	+(10)	5†
HBN5	Nefta	Hippocrepis areolata	+(11)	3ᶲ
HBN6	Nefta	Hippocrepis areolata	+(12)	5†
HBN7	Nefta	Hippocrepis areolata	+(12)	3ᶲ
HBN8	Nefta	Hippocrepis areolata	+(12)	5†
HBN9	Nefta	Hippocrepis areolata	+(13)	3ᶲ
HBN10	Nefta	Hippocrepis areolata	+(10)	5†
HBZ1	Zarzis	Hippocrepis areolata	+(10)	2†
HBZ2	Zarzis	Hippocrepis areolata	+(15)	2†
HBZ3	Zarzis	Hippocrepis areolata	+(14)	3ᶲ
HBZ4	Zarzis	Hippocrepis areolata	+(13)	2†
HBZ5	Zarzis	Hippocrepis areolata	+(12)	2†
HBZ6	Zarzis	Hippocrepis areolata	+(14)	2†
HBZ7	Zarzis	Hippocrepis areolata	+(14)	2†
HBZ8	Zarzis	Hippocrepis areolata	+(13)	5†
HBZ9	Zarzis	Hippocrepis areolata	+(12)	5†
HBZ10	Zarzis	Hippocrepis areolata	+(11)	5†
HBZ11	Zarzis	Hippocrepis areolata	–	1*
HBZ12	Zarzis	Hippocrepis areolata	+(10)	2†
HBZ13	Zarzis	Hippocrepis areolata	+(15)	2†
R. mongolense STM246T= LMG1941T	Mongolia, China	Medicagoruthenica	Nt	6
R. galegae HMBI540T =LMG6214T	Finland	Galegae  orientalis	Nt	7
M. loti ORS664 = LMG6125T	New Zealand	Lotus tenuis	Nt	8
M. mediterraneum ORS2739T=LMG17148T	Spain	Cicer arietinum	Nt	9
S. meliloti ORS665T = LMG6133T	Virginia, USA	Medicagosativa	Nt	3
B. japonicum NZP5549T=LMG6138T	Japon	Glycine max	Nt	10

Note:  STM: collection du laboratoire des Symbioses tropicales et méditerranéennes; HAMBI: Culture Collection of  the Department of Microbiology, University of Helsinki, Helsinki, Finland; LMG: Collection of Bacteria of the Laboratorium voor Microbiologie, Universiteit Ghent, Belgium; NZP: Culture Collection  of the Department for Scientific and Industrial Research, Biochemistry Division, Palmerston North, New Zealand; ORS: Collection, Laboratoire commun de Microbiologie, BP 1386, Dakar, Senegal; ^T type strain. * : isolates grouped in genus Agrobacterium ; †: isolates grouped in genus Rhizobium ; ᶲ: isolates grouped in genus Sinorhizobium, ‡: isolates grouped in genus Mesorhizobium. + Positive test, – no nodulation. Numbers in parentheses indicate the number of nodules per plant, Nt : not tested

Nodulation test. To assess nodulation, seeds were surface-sterilized in 98% sulphuric acid for 30 min and germinated on H₂O-agar plates (0.8%) at 25°C. Seedlings were transferred into vermiculite, inoculated with individual isolates and grown in a growth chamber at 25°C with12-16h photoperiod. Nitrogen-free nutrient solution was used for plant watering (Vincent, 1970). Controls, not inoculated, were included. Four replicates were maintained for each treatment. Four weeks post inoculation, the plants were uprooted and the occurrence of nodulation in each plant was checked.

Phenotypic characterization. All isolates were initially tested for their phenotypic features. For bacterial growth, bacteria were cultivated in 50 ml of YM broth into 250 ml Erlenmeyer flasks and incubated in a gyratory shaker at 180 g and 28°C. Growth was followed by measuring the optical density at 600 nm every 2 h and generation time of each isolate was deduced from the exponential phase of the growth curves.

Growth of the isolates at different temperatures (28, 37, 40, 42, 45°C), the ability to grow in the presence of different NaCl concentrations (1, 2, 3, 4, 5%) and at different pH levels (4, 5, 7, 9, 11) were determined by growth on supplemented YMA as described by Mohamed et al.  (2000).

The modified-YMA medium (Somasegaran and Hoben, 1994) was used to investigate the ability of isolates to use carbohydrates (1% glucose, galactose, fructose and sucrose) and amino-acids (0.1% L- proline, L-arginine, L- tyrosine and L- leucine) as a sole carbon and nitrogen sources respectively. Production of acid or alkali was determined on YMA supplemented with 0 ±0025% (w/v) bromothymol blue as pH indicator. All phenotypic tests were performed in triplicate.

PCR amplification and RFLP analysis of 16S rRNA gene. Total genomic DNA was extracted as described by Mhamdi et al. (2002). Primers fD1 and rD1 (Weisburg et al., 1991), were used for PCR amplification of 16S rRNA gene. PCR was carried out in Gen Amp PCR system 9700 (Applied Biosystems) in a 25 µl containing template DNA extract as described previously by Mahdhi et al. (2012). PCR amplification was analyzed by horizontal 1% (w/v) agarose gel electrophoresis stained with ethidium bromide. The amplified DNA fragments of 16S rRNA gene were digested with RsaI, HinfI, HaeIII, CfoI, NdeII and MspI restriction enzymes (Promega products). The restriction patterns were checked by horizontal 4% (w/v) agarose gel electrophoresis stained with ethidium bromide.

Different 16S rDNA types were designed based upon the combined RFLP patterns obtained from the six enzymes, e.g. isolates is defined as a unique 16S rDNA type if it has one band different from other isolates in the six digestions

Sequencing of 16S rRNA gene. Bacterial genomic DNA extracted according to Mhamdi et al. (2002) was used as templates. For five isolates 16S rDNA gene chosen as representative of different 16S rDNA types, were amplified using universal primers fD1 and rD1 as described above. The PCR products were purified and sequenced using the ABI PRISM BigDye Terminator cycle sequencing kit according to the manufacturer’s protocol and analysed on an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). Sequences were assembled using ChromasPro and were aligned with Clustal X. The acquired sequences were deposited in the GenBank database and were analysed for homologies to related sequences obtained from GenBank. The phylogenetic analyses were performed using mega 3.1 software (Kumar et al., 2001). A neighbour-joining tree was constructed using Kimura two-parameter model (Kimura, 1980) of and support of internal branches was assessed using 1000 bootstrap replications. The GenBank accession numbers for the 16S rRNA gene sequences reported in this paper are KR108303 (ACN5), KR108304 (HBN4), KR108300 (ACM1), KR108301 (ACN1) and KR108302 (ACZ1).

Results

Nodulation test and phenotypic characterisation. A nodulation test was performed for all isolates. Result showed that only two A. corrugatus isolates (ACM1 and ACN10) and one H. areolata isolate (HBZ11) affiliated to Agrobacterium by 16S rRNA gene sequencing analysis (see below) failed to nodulate their host plant of origin. The other isolates formed ten to fifteen nodules per plantlet after four weeks post-inoculation (Table I).

Phenotypically, (Table II) all isolates were acid producers, fast growers (Generation times < 6h). Five A. corrugatus isolates (described by the analysis of 16S rRNA sequences as Mesorhizobium have a generation time between 4 and 6 h. All tested isolates used all tested compounds as sole carbon and nitrogen sources and were able to grow at pHs between 6 and 9, but none of them could grow at pHs 4 and 11.

Table II
Phenotypic characteristics of the isolates

Characteristics	Sinorhizobium isolates	Rhizobium isolates	Mesorhizobium isolates	Agrobacterium isolates
Number of isolates	23	26	5	3
Generation time
G<4h	+	+	–	+
4≤G<6	–	–	+	–
Growth at pH
4	–	–	–	–
9	+	+	+	+
11	–	–	–	–
Acid production	+	+	+	+
Alkali production	–	–	–	–
NaCl tolerance
2%	+	+	+	+
3%	+	+ (20)	+	–
4%	+ (3)	+ (1)	–	–
5%	–	–	–	–
Utilisation of sugars
glucose	+	+	+	+
galactose	+	+	+	+
fructose	+	+	+	+
sucrose	+	+	+	+
Utilisation of amino acids
L-arginine	+	+	+	+
L-proline	+	+	+	+
L-leucine	+	+	+	+
L-tyrosine	+	+	+	+
Growth at temperature
40°C	+	+ (24)	+ (4)	–
42°C	+ (2)	–	–	–
45°C	–	–	–	–

Note: (+) positive growth/ present; (-) no growth/absent; Number in parentheses indicate the number of positive isolates of the total number of isolates tested

The majority of the isolates grew at 28, 37 and 40°C. Only two isolates (ACN1 and ACN4) continued to grow at 42°C, but not at 45°C. Most of the tested isolates tolerated NaCl concentrations from 1 to 3%. Three A. corrugatus isolates (ACN1, ACN4, and ACN13) and one H. areolata isolate (HBZ1) continued to grow in 4% NaCl and none of them tolerated 5% NaCl.

PCR-RFLP analysis of 16S rRNA gene.  The new isolates of 16S ribosomal DNA and reference strains was PCR-amplified and a single band of the expected size of approximately 1500 bp was produced. PCR products were digested with six restriction enzymes RsaI, HinfI, HaeIII, CfoI, NdeII and MspI. Ten 16S rDNA types were distinguished among the 57 isolates and the six reference strains. Each 16S rDNA type comprised 1 to 23 isolates (Table I). Five rDNA types were identified among the new isolates. Types 1, 2, 3 of 16S rDNA included new isolates originating from both A. corrugatus and H. areolata microsymbionts. The type 4 and 5 of 16S rDNA contained only A. corrugatus and H. areolata isolates respectively. Type 3 of 16S rDNA consisted of both new isolates and a reference strain (Sinorhizobium meliloti LMG6133^T).

Sequencing of 16S rDNA. A total of five A. corrugatus and H. areolata isolates representing the five different 16S rDNA types were selected to undergo 16S rDNA gene sequencing. New A. corrugatus and H. areolata strains exhibited 99-100 % 16S rDNA sequence similarity with reference species already described in GenBank. In the reconstructed phylogenetic tree (Fig. 1), strain ACZ1 (Representative of 16S rDNA type 4) was phylogenetically related to Mesorhizobium temperatum CCNWSX0012-2 and to Mesorhizobium sp. LAC831. The two isolates ACN5 and HBN4, representative of 16S rDNA types 2 and 5 respectively, were grouped in Rhizobium branch; with ACN5 strain was associated to Rhizobium sp. STM 394, while HBN4 strain was affiliated related to Rhizobium sp. STM 4037. The sequences of 16S rDNA of the strain ACN1 was closely related to the 16S rDNA sequences of S. meliloti LMG6133^T and Sinorhizobium sp. STM4038, while the strain ACM1 was phylogenetically related to Agrobacterium tumefaciens 2002000903 and Agrobacterium sp. STM4035.

Discussion

Currently, in the rhizobia taxonomy the polyphasic approach, based on phenotypic and genomic criteria is used (Graham et al., 1991; Vandamme et al., 1996; Małek and Sajnaga, 1999). Among the phenotypic features characteristic related to the microorganism ecological niches are considered as the most interesting. In the present studies a collection of 57 isolates was obtained from A. corrugatus and H. areolata root nodules covering four regions of Tunisia and characterized by a polyphasic approach including phenotypic features, PCR-RFLP of 16S rDNA and 16S rDNA sequence analysis. All isolates, except ACM1, ACN10 and HBZ11, re-induce nodules in their host plant. The three non-re-nodulating strains can be considered as opportunistic endophytes as already proposed (Zakhia et al., 2006; Mahdhi et al., 2007; 2012).

Phenotypically (Table II) our results indicate that A. corrugatus and H. areolata isolates showed heterogenic responses to the different phenotypic features. This heterogeneity may contribute the nodulation of legumes in different conditions (Wei et al., 2008). All new isolates are acid producers, fast growers (Generation times < 6h) like Rhizobium, Sinorhizobium and Mesorhizobiun species (Małek and Sajnaga, 1999). All tested isolates are able to use all tested compounds as sole carbon and nitrogen sources. Similar results were reported by Guerrouj et al. (2013) for rhizobia nodulating A. glombiformis in Eastern Morocco. This ability to use a wide range of carbon sources could be beneficial for the bacterial life cycle in the soil and may be related to their high competitiveness in a natural environment. Elkan (1992) reported that carbohydrate sources could be used to differentiate fast-growing rhizobia from the slow-growing bradyrhizobia.

As for salinity, temperature and pH tolerance, our results showed that most of the isolates are able to grow at 3% NaCl, at pHs between 6 and 9 and at high temperature (40°C), except of two of them which were continued to grow at 42°C. These results corroborate our earlier reports on the root nodule bacteria isolated from wild legumes in Tunisia (Mahdhi et al., 2007; 2008; Rejili et al., 2009; Fterich et al., 2011). Similarly, Guerrouj et al. (2013) reported that rhizobial strains associated to A. glombiformis are tolerant to 342 mM NaCl and 40°C in Eastern Morocco. In China, Wei et al. (2003) reported that some rhizobial strains nodulating Astragalus species tolerate only 2% NaCl. The capacity of new isolates to tolerate high temperatures and high NaCl concentrations could be considered a specific adaptation to high soil temperatures and salinity in arid regions as described by Karanja and Wood (1988).

By using the comparative 16S rRNA gene sequence analysis, the new isolates were grouped on the phylogram in the Sinorhizobium, Rhizobium, Mesorhizobium and Agrobacterium genera; with 73% of the new isolates are species within the genera Sinorhizobium and Rhizobium as are many other indigenous legume symbionts from Tunisia (Zakhia et al., 2004; Ben Romdhane et al., 2005; Mahdhi et al., 2008). By 16S rRNA gene sequencing of isolates ACN5 (16S rDNA type 2), HBN4 (16S rDNA type 5) and ACN1 (16S rDNA type 3) are closely related to Tunisian legume nodulating bacteria belonging to Rhizobium sp. STM 394, Rhizobium sp. STM 4037 and Sinorhizobium sp. STM4038 which were isolated from Astragalus cruciatus and Argyrolobium uniflorum growing on the same Tunisian soils (Zakhia et al., 2004; Mahdhi et al., 2008). So, it would now be interesting to test the cross-nodulation capacity of our new isolates on A. cruciatus and A. uniflorum legumes.

It has been previously reported that A. corrugatus is nodulated by strains belonging to the genera Rhizobium, Sinorhizobium, Brdyrhizobium, Mesorhizobium, Agrobacterium and Phyllobacterium (Wei et al., 2003; Gao et al., 2004; Zakhia et al., 2004; Mantelin et al., 2006; Guerrouj et al., 2013; Zheng et al., 2013; Gnat et al., 2014). Our results thus confirm the previous studies showing the large diversity of A. corrugatus rhizobia that belong to Rhizobium, Sinorhizobium, Mesorhizobium and Agrobacterium. However, no Phyllobacterium and Bradyrhizobium were recovered in our results. Others studies (Guerrouj et al., 2013; Gnat et al., 2014) showed that Astragalus glycyphyllos in Poland and A. glombiformis in Morocco were nodulated by rhizobial bacteria belonging only to the genus Mesorhizobium. Among our collection only five A. corrugatus isolates (16S rDNA type 5) were phylogenetically grouped in Mesorhizobium branch, closely related to M. temperatum CCNWSX0012-2 and to Mesorhizobium sp. LAC831 nodulating Lotus creticus from arid regions of Tunisia (Rejili et al., 2009; 2012; 2013). This diversity of rhizobia nodulating Astragalus species may be in relation with climatic and edaphic conditions. Our study confirms that the Astragalus species nodulated by several rhizobial genomspecies can be qualified as promiscuous and that their rhizobia have very diverse genomic and symbiotic gene backgrounds (Zhao et al., 2008; Gnat et al., 2014)

Until now, nodulation of Hippocrepis species has been poorly documented (Zakhia et al., 2004; Muresu et al., 2008). In addition, only two strains were included in these studies. In our collection, H. areolata isolates are identified as Rhizobium, Sinorhizobium and Agrobacterium genera.

Several studies have reported the presence of Agrobacterium strains in nodules of some legumes (de Lajudie et al., 1999; Gao et al., 2001; Liu et al., 2005; Mhamdi et al., 2005; Mrabet et al., 2006), but all them failed to nodulate their original host plants. Here we isolated three Agrobacterim isolates (16S rDNA type 1) that also failed to nodulate their host plants in vitro. The sequences of 16S rDNA of one isolated Agrobacterium isolate (ACM1) is closely related to the 16S rRNA gene sequence of Agrobacterium sp. STM4035, which was isolated by Mahdhi et al. (2008) from the root nodules of the pastoral legume A. uniflorum.

In conclusion, our study is the first report on the characterisation of A. corrugatus and H. areolata in Tunisia. Our investigation showed that LNB originating from nodules of these legumes was genetically diverse and affiliated with Rhizobium, Sinorhizobium, Mesorhizobium and Agrobacterium. Most of A. corrugatus and H. areolata isolates were related to previously described LNB in Tunisian soils. However, rhizobia from other locations that were not covered in this study should be investigated to provide further information about the diversity of bacteria nodulating these legumes in Tunisia

Acknowledgements
This work was supported by the Ministry of High Education and Research Development-Tunisia. The authors thank Dr Philippe de Lajudie, who kindly provided the reference strains

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