Ann Microbiol (2012) 62:61–68 DOI 10.1007/s13213-011-0227-4
ORIGINAL ARTICLE
Legume-nodulating bacteria (LNB) from three pasture legumes (Vicia sativa, Trigonella maritima and Hedysarum spinosissimum) in Tunisia
Mosbah Mahdhi & Amira Fterich & Mokhtar Rejili & Ignacio David Rodriguez-Llorente & Mohamed Mars
Received: 21 September 2010 /Accepted: 7 February 2011 /Published online: 22 February 2011 # Springer-Verlag and the University of Milan 2011
Abstract Sixty-one bacterial isolates were recovered from Introduction surface-sterilized root nodules of Vicia sativa, Trigonella maritima and Hedysarum spinosissimum plants growing in Legume-nodulating bacteria (LNB) are Gram-negative soil two arid Tunisian soils. The natural nodulation resource of bacteria that fix nitrogen after becoming established inside root these legumes, prospected from the two sites, was investi- nodules of legumes. In the last few years, a large diversity of gated. The occurrence of nodulation and the morphology of LNB has been revealed, which has caused considerable the nodules were observed. The isolates were examined by changes in the taxonomy of these bacteria. The current phenotypic characterization and 16S rDNA analysis. taxonomy of LNB reveals a wide diversity at the genus, Among the 61 isolates that were screened, the majority species and intraspecies levels. Most of these bacterial species (92%) were fast-growing rhizobia. Twenty-eight strains are in the Rhizobiaceae family in the α-class of Proteobacteria: tolerated high concentration of salt (3% NaCl) and grew at Rhizobium, Azorhizobium, Sinorhizobium (syn. Ensifer), temperatures up to 40°C. PCR restriction fragment length Mesorhizobium, Bradyrhizobium, Methylobacterium (Jaftha polymorphism (PCR-RFLP) and 16S rRNA gene sequencing et al. 2002; Jourand et al. 2004), Devosia (Rivas et al. 2003), revealed that the majority of the isolates belonged to the Blastobacter (Van Berkum and Eardly 2002), Ochrobactrum genera Rhizobium (54%) and Sinorhizobium (42%). Five H. (Trujillo et al. 2005)andPhyllobacterium (Valverde et al. spinosissimum isolates failed to nodulate their host plant, and 2005;Mantelinetal.2006). Moreover, about eight rhizobial were affiliated to Pseudomonas and Kocuria genera. This species within two genera of the β-class of Proteobacteria study is the first report that describes bacteria of genus (Burkholderia and Ralstonia)havebeenreported(Moulinet Kocuria occupying root nodules of legumes to the best of al. 2001;Chenetal.2001, 2006, 2008; Leelahawonge et al. our knowledge. 2010; Shiraishi et al. 2010). On the other hand, Agrobacterium strains have been isolated from nodules of many legume Keywords Rhizobia . PCR-RFLP. Nodulation . species (Gurtler et al. 1991; Liu et al. 2005;Mahdhietal. Gammaproteobacteria . Sequencing 2008), but no definitive explanation of the presence of these bacteria inside nodules could be demonstrated. In addition, bacteria from the γ-class of Proteobacteria have also been M. Mahdhi (*) : A. Fterich : M. Rejili : M. Mars reported (Benhizia et al. 2004;Muresuetal.2008; Laboratoire de Biotechnologies Végétales Appliquées à Leelahawonge et al. 2010; Shiraishi et al. 2010). l’Amélioration des Cultures, Université de Gabès, Plants of the genus Vicia, Trigonella and Hedysarum are Faculté des Sciences de Gabès, annuals, which play an important role for forage and Cité Erriadh Zrig, 6072 Gabès, Tunisia medicinal products. Although nodulation of these legumes e-mail: [email protected] has been reported, a detailed description of the taxonomy of the rhizobial symbionts has not been made. Legumes from I. D. Rodriguez-Llorente genera Vicia are commonly nodulated by Rhizobium Departamento de Microbiología y Parasitología, Facultad de Farmacia, leguminosarum bv. viciae (Jordan 1984; Laguerre et al. Sevilla, Spain 2003; Mutch et al. 2003; Slattery et al. 2004; Mutch and 62 Ann Microbiol (2012) 62:61–68
Young 2004; Moschetti et al. 2005). Although, Trigonella Bacterial isolates and reference strains plants mainly nodulate with Sinorhizobium meliloti (Roumiantseva et al. 2002; You et al. 2008), strains Sixty-one isolates and three reference strains (Table 1), belonging to the genera Bradyrhizobium (Pandey et al. representing different rhizobial species belonging to 2004)andRhizobium (Wang et al. 2006; Hou et al. 2009) Rhizobium and Sinorhizobium genera, were used. Rhizo- have also been isolated from two Trigonella species in bial bacteria were isolated from naturally occurring root China. The genus Hedysarum L. comprises about 100 nodules collected in two arid soils of Tunisia: National species, of herbaceous legumes, widely distributed in the park of Bouhedma (34°42′47″N, 9°28′27″E) and Matmata Mediterranean, temperate Europe, North and South Africa, 33°41′N, 10°23′E). For rhizobia isolation, nodules were Asia Minor, Siberia, North America from Arizona into rehydrated in sterile water and surface sterilised by Alaska and the Arctic regions of Canada. Only 19 species immersionin95%ethanolfor30sand0.1%mercuric of Hedysarum are recorded as being nodulated and root- chloride for 2 min. Each nodule was rinsed ten times in nodule bacteria have only been purified and authenticated sterile water. A 100-μl aliquot of the last washing solution from a few of these. Since previous studies on biological was checked for sterility by inoculation on YMA agar nitrogen fixation in the genus Hedysarum focused mainly plates and incubation. Only nodules resulting in a sterile on H. coronarium, there is little information regarding final washing liquid were further considered for bacterial root-nodulating bacteria associated with other Hedysarum isolation. Nodules were then individually squashed and species such as H. spinosissimum. Previous researches streaked on plates containing YMA agar (Vincent 1970). showed that Hedysarum species have been reported to be After incubation for 5 days at 28°C, single colonies were nodulated by Mesorhizobium (Kishinevsky et al. 2003; selected and transferred on to YEMA plates to ascertain Safronova et al. 2004), Rhizobium (Squartinietal.2002; purity. Pure cultures of the isolates or strains were Hung et al. 2005), Bradyrhizobium (Hung et al. 2005)and maintained on YEM agar slants at 4°C or in 25% glycerol Sinorhizobium (Zakhia et al. 2004). In addition, strain at −80°C. SH199 isolated from Hedysarum scoparium in China was grouped as Agrobacterium tumefaciens (Wei et al. 2008). Phenotypic and nodulation tests Another study showed that some isolates from the nodules of Hedysarum species belong to the γ-class of All isolates were observed for colour and colonoy mor- Proteobacteria (Benhizia et al. 2004;Muresuetal.2008). phology after growth on YMA plates for 48 h at 28°C. In Tunisia, there are no reports about rhizobial symbionts Generation time was determined by inoculation in 50 ml of from Hedysarum species, and only one Sinorhizobium YM broth into 250-ml Erlenmeyer flasks and incubation in strain has been isolated from Hedysarum carnosum a gyratory shaker at 180g and 28°C. Growth was checked (Zakhia et al. 2004). by measuring the optical density at 600 nm every 2 h. The Considering the importance of Vicia sativa, Trigonella generation time was deduced from the exponential growth maritima and Hedysarum spinosissimum in forage produc- phase. The growth temperature ranges (15, 28, 35, 37, 40, tion and the insufficient study on the diversity of rhizobia 42°C), the ability to grow in the presence of NaCl (1, 2, 3, associated with these three legumes, the aim of this study is 4%) and at different pH (4, 5, 6, 7, 9, 10, 12) were tested on to analyse, using both phenotypic and genotypic methods, YMA plates as described by Mohamed et al. (2000). the taxonomic diversity of 61 nodule isolates from these Each plate was divided into 12 equal sectors, and each legumes grown in two arid areas of Tunisia. Natural sector was then inoculated with 10 μlofexponentialphase nodulation of these three legumes is also investigated and growth cultures of the test strain. Acid and alkali is reported for the first time. production were determined in YEM agar medium with bromothymol blue indicator (0.0025%, w/v). All pheno- typic tests were done in triplicate. Materials and methods All isolates were tested for their ability to re-nodulate their host plant. Inoculation and seed treatment were Occurrence of nodulation performed as described by Mahdhi et al. (2008). Seeds were scarified with 95% sulphuric acid for 10 min. Spontaneous nodulation of the three plant species pro- Germinated seedlings were aseptically transferred to indi- spected in this study is reported from their sites of origin. vidual pots of about 200 ml containing vermiculite and The intensity of nodulation (number of nodules per plant) sterilised nitrogen-free nutrient solution (Vincent 1970). and the morphology of nodules (shape and colour) were The pots were placed in a growth chamber at 25°C during investigated visually. Twelve plants were considered at the day and 18°C at night, with a 16-h photoperiod and 60– each site. 70% relative humidity. Two days later, each seedling was Ann Microbiol (2012) 62:61–68 63
Table 1 Isolates and reference strains used in this study and their relevant characteristics
Isolates and Other Host plant Site of origin 16S Isolates and Host plant Site of origin 16S reference strains designation rDNA reference rDNA type strains type
VB1 V. sativa Bouhedma Park, 1 TB6 T. maritima Bouhedma Park, 2 Tunisia Tunisia VB2 V. sativa Bouhedma Park 1 TB7 T. maritima Bouhedma Park 2 VB3 V. sativa Bouhedma Park 1 TB8 T. maritima Bouhedma Park 2 VB4 V. sativa Bouhedma Park 1 TB9 T. maritima Bouhedma Park 2 VB5 V. sativa Bouhedma Park 1 TB10 T. maritima Bouhedma Park 2 VB6 V. sativa Bouhedma Park 1 TB11 T. maritima Bouhedma Park 2 VB7 V. sativa Bouhedma Park 1 TB12 T. maritima Bouhedma Park 2 VB8 V. sativa Bouhedma Park 1 TB13 T. maritima Bouhedma Park 2 VB9 V. sativa Bouhedma Park 1 TB14 T. maritima Bouhedma Park 2 VB10 V. sativa Bouhedma Park 1 TB15 T. maritima Bouhedma Park 2 VB11 V. sativa Bouhedma Park 1 TB16 T. maritima Bouhedma Park 2 VB12 V. sativa Bouhedma Park 1 TB17 T. maritima Bouhedma Park 2 VB13 V. sativa Bouhedma Park 1 TB18 T. maritima Bouhedma Park 2 VB14 V. sativa Bouhedma Park 1 TB19 T. maritima Bouhedma Park 2 VB15 V. sativa Bouhedma Park 1 TB20 T. maritima Bouhedma Park 2 VB16 V. sativa Bouhedma Park 1 TB21 T. maritima Bouhedma Park 2 VB17 V. sativa Bouhedma Park 1 TB22 T. maritima Bouhedma Park 2 VB18 V. sativa Bouhedma Park 1 TB23 T. maritima Bouhedma Park 2 VB19 V. sativa Bouhedma Park 1 TB24 T. maritima Bouhedma Park 2 VB20 V. sativa Bouhedma Park 1 TB25 T. maritima Bouhedma Park 2 VB21 V. sativa Bouhedma Park 1 TB26 T. maritima Bouhedma Park 2 VB22 V. sativa Bouhedma Park 1 TB27 T. maritima Bouhedma Park 2 VB23 V. sativa Bouhedma Park 1 TB28 T. maritima Bouhedma Park 2 VB24 V. sativa Bouhedma Park 1 TB29 T. maritima Bouhedma Park 2 TB1 T. maritima Bouhedma Park 2 TB30 T. maritima Matmara, Tunisia 2 TB2 T. maritima Bouhedma Park 2 HM1 H. spinosissimum Matmara 2 TB3 T. maritima Bouhedma Park 2 HM2 H. spinosissimum Matmara 4 TB4 T. maritima Bouhedma Park 2 HM3 H. spinosissimum Matmara 4 TB5 T. maritima Bouhedma Park 2 HM4 H. spinosissimum Matmara 4 S. meliloti NZP4027T, LMG6133T Medicago sativa Virginia, USA 2 HM5 H. spinosissimum Matmara 4 ORS665T Sinorhizobium medicae LMG16580, Medicago. Syria 3 HM6 H. spinosissimum Matmara 5 M102, ORS504 HAMBI1809 truncatula R. leguminosarum bv. VF39SM 1 HM7 H. spinosissimum Matmara 2 viciae ORS639
Names of strains reflect their host plant and site of isolation: TB Trigonella maritima isolates from Bouhedma Park, Tunisia; HM Hedysarum spinosissimum isolates from Matmata, Tunisia; VB Vicia sativa isolates from Bouhedma Park, Tunisia; 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 inoculated with 1 ml of culture of the appropriate rhizobial (2002). Bacteria were grown on TY agar slopes at 28°C for strain (approximately 109 cells). Uninoculated plants served 48 h and suspended in sterile water. A volume, as control treatment. Three replicates were maintained for corresponding to 200 μl matching a DO620 nm=1, was each treatment. Thirty days later, plants were examined for centrifuged. The cell pellet was resuspended in Tris-HCl root nodulation. (10 mM, pH 8.3) and 20 μl of proteinase K (1 mg/ml). Cells were incubated overnight at 55°C and then 10 min PCR-RFLP of 16S rRNA gene in boiling water. Bacterial Cell preparations were stored at −20°C until use and a volume of 5 μl was used for PCR Bacterial genomic DNA from each strain was extracted amplification as emplate DNA. Two primers FGPS 6 (5′- according to the methodology described by Mhamdi et al. GGAGAGTTAGATCTTGGCTCAG-3′) and FGPS 1509 64 Ann Microbiol (2012) 62:61–68
(5′-AAGGAGGGGATCCAGCCGCA-3′) (Weisburg et al. Results 1991) were used for PCR amplification of 16S rRNA gene. PCR reaction was carried out in a final volume of Occurrence of nodulation and nodule morphology
25 μl containing: 1× PCR buffer, 2.5 mM of MgCl2, 0.02 mM of each dNTP, 1 U of Taq DNA polymerase, Natural nodulation of the three plant species prospected in
0.1 μMofeachprimer,6μlofH2Oand5μl of template this study is reported. Results show that V. sativa was DNA (100 ng). The amplifications were performed in a strongly nodulated (52 nodules/plant). The lowest number Thermocycler (Gene Amp PCR System 9700; Applied of nodules (12 nodules/plant) was observed for H. Biosystems) and programmed for an initial denaturation of spinosissimum. The external colour of the nodules is white 3minat95°C,followedby35cyclesof1minat94°C, or brown. The size of the nodules with globular shapes 1 min at 55°C and 2 min at 72°C, and a final extension of varied from 0.2 to 0.3 cm, and could exceed the diameter of 2 min at 72°C. PCR products were checked by electro- the root of their host plant. phoresis on 1% agarose gel. Aliquots (7 μl) of PCR products were digested with each of the following Phenotypic properties enzymes: RsaI, NdeII, AluI, HinfI, MspI and TaqI. Restriction DNA was analysed by horizontal electropho- Sixty-one isolates investigated in this study (Table 1) resisin4%agarosegelat4Vcm−1. Each restriction band were purified from root nodules of three forage legumes was considered as a unit feature and was scored as 1 (V. sativa, T. maritima and H. spinosissimum)growingon (present) or 0 (absent) for all strains. The similarity two arid soils of Tunisia. A nodulation test was performed coefficient (Ssm) was calculated as Ssm ¼ m=ðm þ nÞ, for all the isolates. Results showed that only five H. where m is the number of matched bands and n is the spinosissimum isolates, which were classified as γ-class of number of mismatched bands. A dendrogram was con- Proteobacteria by 16S rRNA gene sequencing analyses structed from the similarity matrix using the unweighted (see below) failed to nodulate their host plant of origin. pairgroupmethodwithaverages(UPGMA)(Sneathand Phenotypically, the majority of the isolates were acid Sokal 1973). producers (colonies change colour after 72 h at 28°C to yellow) and fast growing with average mean generation Sequencing of 16S rDNA time of 3 h. Most of the isolates were able to grow at pH between6and9,butonlytwoisolates(VB15andVB13) The 16S rRNA genes of three H. spinosissimum grow at pH 4 (Table 2). The tolerance to NaCl and isolates, chosen as representatives for each 16S rDNA resistance to temperature were variable among the studied pattern, were amplified under the conditions described isolates: most isolates were able to grow only at 2% NaCl above. PCR products were run on a 1% agarose gel, band and at 37°C while the majority of T. maritima isolates was excised and DNA purified using CTB DNA Extrac- grew well in YEM broth with 3% NaCl and at 40°C but tion Mini Kit (iNtRON Biotechnology, Korea). Two not with a 4% NaCl concentration and at 42°C. A summary of forward primers (FGPS6 and 16S-370f) and one reverse some phenotypic properties is described in Table 2. primer (FGPS1509) were used to obtain the complete gene sequence. 16S rRNA gene cycle sequencing was 16S-RFLP analysis performed using the ABI PRISM BigDye Terminator cycle sequencing kit according to the manufacturer’s The 16S rDNA of all the isolates and reference strains was protocol and analysed on an ABI PRISM 310 Genetic amplified, resulting in a characteristic single band of Analyzer (Applied Biosystems). The sequences obtained 1500 bp. Restriction analysis was performed by using six were deposited in the GenBank database and were endonucleases. The analysis revealed three to five restric- compared with related sequences from the database. The tion patterns per enzyme. Five 16S rDNA types were sequences were aligned using the programs in the distinguished among the 61 isolates and the reference package of Clustal X and Genedoc software. The strains. Each 16S rDNA type comprising 1–33 isolates phylogenetic analyses were performed using mega 3.1 (Table 1). All V. sativa isolates had 16S rDNA type software (Kumar et al. 2001). A neighbour-joining tree identical to that of the reference R. leguminosarum bv. was reconstructed and bootstrapped with 1000 replica- viciae ORS639 (16S rDNA type 1). 16S rDNA type 2 tions of each sequence using Kimura two-parameter included 32 new isolates, originating from T. maritima (30 model (Kimura 1980) of evolution. The GenBank acces- isolates) and H. spinosissimum (2 isolates). These isolates sion numbers for the 16S rRNA gene sequences reported share identical PCR- 16S rDNA RFLP pattern with in this paper are HM2 (GU384322), HM6 (GU384323) Sinorhizobium meliloti type strain (ORS665T). Four H. and HM1 (GU384324). spinosissimum isolates (HM2, HM3, HM4, and HM5) have Ann Microbiol (2012) 62:61–68 65
Table 2 Phenotypic characteris- tics of the isolates Characteristics V. sativa isolates T. maritima isolates H. spinosissimum isolates
Number of isolates 24 30 7 Generation time in YEM medium GT≤3 h + (20) + (20) + (2) 3
The N2-fixing leguminous plants are key components of the Hedysarum species, Trigonella foenum-graecum and Vicia natural succession in semi-arid Mediterranean ecosystems angustifolia. Moschetti et al. (2005)reportedthat19 because these plant species, upon establishing rhizobial isolates from the nodules of several Vicia species (V. symbioses, constitute a fundamental source of N input to hybrida, V. sativa, V. faba and V. villosa) in central and the ecosystem (Zahran 2001). Spontaneous nodulation of southern Italy were not capable of tolerating salt concen- 66 Ann Microbiol (2012) 62:61–68 trations above 0.1%, and only two of them tolerated 2% (w/ ments. No reports have been published relating to Kocuria– w) NaCl. These results prove that the phenotypic properties plant interactions or to their nitrogen fixing ability of rhizobial strains, such as temperature resistance and In our collection, four H. spinosissimum isolates are NaCl tolerance, can be influenced by climatic and edaphic identified as the genus Pseudomonas by PCR-RFLP and conditions. Many T. maritima isolates identified as S. 16S rRNA gene sequencing. Pseudomonas is a genus of meliloti tolerate high temperatures (40°C) and NaCl gamma Proteobacteria. Pseudomonas species are a group concentrations (up 3%, w/v). These isolates may be of bacteria found commonly in soil and other natural candidates for Trigonella species inoculation in arid soils. environments. Some Pseudomonas species have been These appropriate native rhizobia resistant to temperature reported to be endophytic of legume (Elvira-Recuenco and salinity would guarantee root nodulation and enhance and Van Vuurde 2000) and also non-legume plants (Kovacs plant performance. However, selection of these rhizobia et al. 1999; Lodewyckx et al. 2002). Previous studies would need to take into consideration not only their N2- (Benhizia et al. 2004; Muresu et al. 2008) showed that fixing capacity but also their competitive ability against Pseudomonas strains could be isolated from root nodules of native rhizobia. Superior N2-fixing strains have to outcom- several Hedysarum species. On the other hand, nitrogen pete native rhizobia and occupy a significant proportion of fixation has been reported for some species of Pseudomonas the nodules (Rengel 1992). (Young 1992;Desnouesetal.2003). Shiraishi et al. (2010) It has been previously reported that rhizobial strains reported that Pseudomonas strains formed nodules on black from several Vicia sp. were grouped with R. leguminosarum locust and also developed differentiated nodule tissue. bv. viciae (Laguerre et al. 2003; Slattery et al. 2004; Mutch Leelahawonge et al. (2010) proposed a bacterium related to and Young 2004; Moschetti et al. 2005). Alvarez-Martinez Pseudoalteromonas as a new symbiont of the medicinal et al. (2009) suggest a world distribution of strains from R. legume Indigofera tinctoria. These two Gammaproteobacteria leguminosarum together with V. sativa and V. faba seeds. (Pseudoalteromonas and Pseudomonas) are found to be true Similar results were found for all V. sativa isolates symbionts of their legumes as they harbour nifH and nodC investigated in our study. genes. These two major symbiotic genes have high similarities PCR-RFLP analysis showed that all T. maritima isolates with those of rhizobial species. Lateral gene transfer from have their 16S rDNA type identical to S. meliloti LMG6133T Rhizobiales is suggested as one of the mechanisms explaining (Table 1). This finding confirmed recent reports on root the acquisition of endophytic interactivity with leguminous nodule bacteria of other Trigonella sp. (You et al. 2008). plants. In our study, we could not find evidence of nodulation However, strains belonging to the genera Bradyrhizobium capacity among Pseudomonas and Kocuria isolates, so these (Pandey et al. 2004)andRhizobium (Hou et al. 2009)have isolates can be considered as opportunistic endophytic also been isolated from T. foenum-graecum and Trigonella bacteria as already reported by Zakhia et al. (2006)and archiducis-nicolai,respectively. Mahdhi et al. (2007, 2008), and this explanation confirms the Full-length 16S rDNA sequencing grouped one H. nodulation failures of these isolates. Here, we did not screen spinosissimum (HM6) to the genus Kocuria.Micro- these isolates for symbiotic genes. Therefore, further inves- organisms of the genus Kocuria are Gram-positive, belong- tigations on the nodulation gene properties of Pseudomonas ing to the family Micrococcaceae, Their normal habitats and Kocuria would be helpful to understand the nature of include mammalian skin (Savini et al. 2010; Tsai et al. bacterial interaction. Such investigations are necessary before 2010), soil (Li et al 2006), the rhizosphere (Takarada et al. the status of these isolates as nodules symbionts could be 2008), fermented foods, and freshwater and marine sedi- considered. The non-nodulation in vitro of H. spinosissimum
Fig. 1 Phylogenetic tree of 16S rDNA showing the relationships among the isolates and the related bacterial species. Significant bootstraps (>80%) are indicated as percentages (1000 replica- tions). Sequence accession numbers are listed in parentheses Ann Microbiol (2012) 62:61–68 67 by some isolates may also indicate that these legumes are not Chen WM, De Faria SM, Chou JH, James EK, Elliott GN, Sprent JI, the natural hosts of these strains. It would now be interesting Bontemps C, Young JP, Vandamme P (2008) Burkholderia sabiae to demonstrate their capacities to induce nodule development sp. nov., isolated from root nodules of Mimosa caesalpiniifolia.Int J Syst Evol Microbiol 58:2174–2179 on other wild legumes grown on the same Tunisian soils. Desnoues N, Lin M, Guo X, Ma L, Carreño-Lopez R, Elmerich C Using two phylogenetic methods (16S rDNA PCR-RFLP (2003) Nitrogen fixation genetics and regulation in a Pseudomonas and sequencing), two H. spinosissimum isolates (HM1 and stutzeri strain associated with rice. Microbiolgy 149:2251– HM7) were grouped with Sinorhizobium. The HM1 isolate 2262 Elvira-Recuenco M, Van Vuurde JWL (2000) Natural incidence of was similar to the Sinorhizobium meliloti Lse-2 strain endophytic bacteria in pea cultivars under field conditions. Can J originating from Lotus sp. in the Canary Islands (Fig. 1). Microbiol 46:1036–1041 This finding supported results reported by Zakhia et al. Gurtler V, Wilson VA, Mayall BC (1991) Classification of medically (2004) with Sinorhizobium strain isolated from H. carnosum important clostridia using restriction endonuclease site differ- ences of PCR-amplified 16S rDNA. J Gen Microbiol 137:2673– in Tunisia. However, Kishinevsky et al. (2003) found that 2679 H. spinosissimum isolates were closely related to both Hou BC, Wang ET, Ying LJ, Jia RZ, Chen WF, Gao Y, Don RJ, Chen Mesorhizobium loti and M. ciceri, sharing 97% identity WX (2009) Rhizobium tibeticum sp. nov., a symbiotic bacterium with each species. isolated from Trigonella archiducis-nicolai Vassilcz. Int J Syst Evol Microbiol 59:3051–3057 In summary, this study is the first report on the Hung MH, Bhagwath AA, Shen FT, Devasya RP, Young CC (2005) characterisation of H. spinosissimum microsymbionts in Indigenous rhizobia associated with native shrubby legumes in Tunisia. Our results support the presence of the γ-class of Taiwan. Pedobiologia 49:577–584 Proteobacteria in root-nodules of Hedysarum species. Two Jaftha JB, Strijdom BW, Steyn PL (2002) Characterization of pigmented methylotrophic bacteria which nodulate Lotononis Sinorhizobium isolates were also isolated from this legume. bainesii. 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