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.