Ann Microbiol (2010) 60:541–547 DOI 10.1007/s13213-010-0084-6 ORIGINAL ARTICLE Genetic diversity and salt tolerance of Sinorhizobium populations from two Tunisian soils Darine Trabelsi & Alessio Mengoni & Mohammed Elarbi Aouani & Marco Bazzicalupo & Ridha Mhamdi Received: 24 March 2010 /Accepted: 9 June 2010 /Published online: 30 June 2010 # Springer-Verlag and the University of Milan 2010 Abstract Leguminous plants are promising pioneer-species Introduction for colonization of marginal areas and soils undergoing desertification. They derive part of their colonization abilities Root-nodule bacteria, known as rhizobia and belonging to from their symbiotic interaction with nitrogen-fixing bacteria different genera of alpha- and beta-Proteobacteria, form known as rhizobia. In this work, we analyzed total and nitrogen-fixing symbioses with legumes. The agricultural culturable rhizobium communities from two Tunisian soils in value of rhizobia has been long been widely recognized, as close proximity to each other: one non-salty and cultivated they improve soil fertility through nitrogen fixation and and the other uncultivated and salty. The taxonomic diversity reduce the need for application of nitrogenous fertilizers. of the Rhizobiaceae family, evaluated by terminal restriction Within the rhizobia, members of the alpha-Proteobacteria fragment length polymorphism (T-RFLP) on total soil DNA, family Rhizobiaceae are particularly important in nitrogen- revealed a high diversity of ribotypes and soil-based fixing symbioses throughout the world (Vance 1998), differentiation of the communities. We then used PCR-RFLP providing about 90 million tons of fixed nitrogen per year of the intergenic space and salt tolerance to characterize the worldwide. The genus Sinorhizobium (syn. Ensifer) and, in genetic and phenotypic polymorphism of 150 Sinorhizobium particular, strains belonging to the species Sinorhizobium isolates trapped on Medicago truncatula. In the salty soil, meliloti and Sinorhizobium medicae, are ubiquitous free-living two different species, Sinorhizobium meliloti and Sinorhi- soil-bacteria that also specifically form symbiotic nitrogen- zobium medicae, were trapped; by contrast, only isolates of fixing nodules on Medicago, Melilotus and Trigonella. S. meliloti were trapped from the agricultural soil. Moreover, However, it was also reported that S. meliloti can be isolated isolates from the salty soil were more tolerant to NaCl, and from many other legume genera, i.e., Phaseolus vulgaris strains growing up to 1 M NaCl were found. No relation- (Mhamdietal.2002;Mnasrietal.2007), Cicer arietinum ships between genotypic profiles and salt tolerance pheno- (Ben Romdhane et al. 2007), Argyrolobium uniflorum, Lotus types were found. creticus, Lotus roudairei, Ononis natrix, Retama raetam, Genista saharae, Acacia tortilis, Hedysarum carnosum, Keywords Diversity. Medicago truncatula . Sinorhizobium Hippocrepis bicontorta (Zakhia et al. 2004;Mahdhietal. meliloti . Sinorhizobium medicae . Salinity. T-RFLP 2007), which are of special interest in the agro-pastural systems of sub-arid regions. Among the plants nodulated by : : sinorhizobia, several are able to grow on marginal soils and on D. Trabelsi (*) M. E. Aouani R. Mhamdi sub-arid habitats. In particular, species of the genus Medicago Laboratoire des Légumineuses, are present as pioneer species in many saline soils and in soils Centre de Biotechnologie de Borj-Cedria, BP 901, Hammam-Lif 2050, Tunisia undergoing desertification (Subbian et al. 2000). This is highly e-mail: [email protected] relevant as nearly 40% of the world’s land surface can be : recorded as having potential salinity problems (Zahran 1999). A. Mengoni M. Bazzicalupo Actually, salt stress is one of the major environmental Department of Evolutionary Biology, University of Firenze, via Romana 17, stress-conditions that adversely affect legume production 50125 Florence, Italy (Shamseldin et al. 2006) and distribution of rhizobia in 542 Ann Microbiol (2010) 60:541–547 soil and rhizosphere of plants in arid and semiarid regions (for details on soils and sampling procedures, see Trabelsi (Tate 1995). Salt-tolerant rhizobia have been isolated from et al. 2009). The enumeration of sinorhizobia was deter- various crops and wild legumes. Some of these rhizobia mined by the most probable number (MPN) method are tolerant to high levels of salt—up to 1.8 M NaCl—and (Vincent 1970)usingMedicago truncatula, Medicago have the potential to form a successful symbiosis with sativa and Medicago ciliaris (Table 1). legumes under salt stress (Bernard et al. 1986; Lal and Khanna 1995;Zahran1999; Payakapong et al. 2006; Terminal restriction fragment length polymorphism analysis Mnasri et al. 2007). The study of symbiosis between of the Rhizobiaceae community in soil rhizobia and plants indeed represents a major challenge in basic and applied microbiology aimed at improving Total DNA was extracted from soil samples as previously legume yield, as well as their cultivation as forage, described (Trabelsi et al. 2009). The 16S rRNA genes were bioenergy crops and for restoration of marginal and amplified directly from total soil DNA using the Rhizobia- salinized lands (Provorov and Tikhonovich 2003). ceae specific reverse primer (RHIZ primer; Tom-Petersen et It is a common notion that genetic diversity of rhizobia is al. 2003) and the universal forward primer 27f (Lane 1991). an advantageous trait of these soil communities, extending The forward universal primer 27f was labelled with FAM (6- the range of potential host plants and preventing the fluoro-carboxyfluorescein). Terminal restriction fragment selective effect of environmental stresses (Palmer and length polymorphism (T-RFLP) analysis was performed with Young 2000; Zhang et al. 2001). Population genetics CfoI, RsaIandMspI restriction enzymes. investigations and evaluation of salt tolerance of rhizobia Labelled restriction products from forward primer were from saline soils are of special interest. In particular, the analyzed by capillary electrophoresis on an ABI310 linkage between salt tolerant phenotypes and genotypes is Genetic Analyzer (Applied Biosystems, Foster City, CA) still not clear, as we do not know if a highly adaptive using ROX 500 as size standard. DNA fragments from 35 phenotype such as salt tolerance is harbored by particular to 500 bp were considered for profile analysis. Putative genetic groups in the population or if it is a widespread taxonomic assignment of terminal-restriction fragments feature not associated to particular genotypes (Talebi Bedaf (TRFs) was done by using MiCA3 server application (Shyu et al. 2008). Recently, we have undertaken the analysis of et al. 2007). Analysis of T-RFLP profiles of the Rhizobia- two soils at a site in Soliman (Northern Tunisia) that show ceae family was performed following previously described remarkable differences with respect to cultivation and soil procedures (Mengoni et al. 2007). Derivative T-RFLP physico-chemical parameters, one being a salty and profiles of the different enzymes were combined together neglected soil, the other a cultivated soil (Trabelsi et al. and a binary vector (1-0), in which presence or absence of 2009). Previous analyses showed the presence of a large bacterial taxonomic diversity in the two soils (by both Table 1 Physico chemical characteristics and sinorhizobia population cultivation and culture-independent analyses) and of several density in the two soils from Soliman (North Tunisia). EC Electrical bacterial isolates tolerant to high NaCl concentrations (up to conductivity, MPN most probable number 1 M). Interestingly, among these isolates, representatives of the genus Rhizobium were found (Trabelsi et al. 2009). The Agricultural Saline soil soil aims of the present work were: (1) to estimate the genetic diversity of bacteria belonging to the Rhizobiaceae family pH 8.46±0.13 8.93±0.13 in the cultivated and the saline soil; (2) to estimate the Moisture content (%) 32±1 54±4 genetic diversity of the Sinorhizobium populations in the EC (mmho/cm)a 0.7±0.11 4.66±1.72 same soils; and (3) to isolate and characterize salt-tolerant Organic matter (%) 0.75±0.4 1.1±0.16 sinorhizobia and to check for the presence of a relationship Carbon (%) 0.53±0.23 0.66±0.06 between salt tolerance and genotypic groups. Texture Sandy Clay Sinorhizobia population density (MPN)b Materials and methods Medicago ciliaris (8.12) 5.6 × 10 3 3.3 × 104 Medicago truncatula (TN8.20) 3.3 × 10 3 3.3 × 104 Soil sampling Medicago sativa (Gabes) 0.1 × 103 0.3 × 104 a 1 mmho/cm=1 dS/m Two different soils in close proximity in Soliman (longi- b ′ ″ ′ ″ Homogenized soil samples (10 g) were used for MPN estimates by the tude, 10°29 30 E; latitude, 36°41 47 N) were sampled plant infection count in ten-fold dilution series using four replicates (Table 1) Three replicates were sampled across each soil, (Vincent 1970). The 95% fiducial limits of the MPN estimates are 3.8-fold at a distance of about 35 m between each sampling point more or less than the indicated values (Brockwell 1980) Ann Microbiol (2010) 60:541–547 543 peaks was scored. The vectors of binary profiles were then of 16S-23S rDNA intergenic spacer (IGS) using two compared to compute the similarity matrix using the restriction enzymes, AluI and TaqI, as described by Biondi Jaccard index as implemented in the software NTSYSpc et al. (2003). Restriction products were separated by 2.02 (Rohlf 1990). The matrix of Jaccard similarity values agarose gel (2.5% w/v) electrophoresis