Ann Microbiol (2010) 60:541–547 DOI 10.1007/s13213-010-0084-6

ORIGINAL ARTICLE

Genetic diversity and salt tolerance of 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 are promising pioneer-species Introduction for colonization of marginal areas and soils undergoing desertification. They derive part of their colonization abilities Root-nodule , known as and belonging to from their symbiotic interaction with nitrogen-fixing bacteria different genera of alpha- and beta-, 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 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 truncatula. In the salty soil, meliloti and , 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. . 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 (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 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 in TAE buffer at was then used to construct an unweighted pair group 10 Vcm−1 for 1.5 h and stained in a 1 μgml−1 (w/v) method with arithmetic mean (UPGMA) dendrogram using ethidium bromide solution. the module present in the NTSYSpc 2.02 (Rohlf 1990).

Sampling of Medicago truncatula nodulating sinorhizobia Results and discussion

Medicago truncatula was used as trapping plant because it Total rhizobial community is a predominant natural plant growing in Soliman soil. The same local cultivar as that growing in the sampling site was To investigate and compare the diversity of the Rhizobia- used (M. truncatula line 8.3). Medicago truncatula seeds ceae in the two soils, we first used rhizobium specific were sterilized, scarified and planted in 500 ml aseptic T-RFLP analysis of the 16S rRNA gene fragments. The plastic pots. Each soil type used was a mix of three primer pair 27f/RHIZ was described to target 16S rRNA replicated samples of the same soil. Five plants were used from Rhizobium strains but not from Bradyrhizobium for each soil, and were grown in a greenhouse under natural strains (Tom-Petersen et al. 2003). We also performed a light. At flowering, plants were harvested and the nodules BLAST search and identified target regions in the 16S were collected. Plants showed an average of 25±3.6 rRNA genes of the entire family of Rhizobiaceae, including nodules /plant from the cultivated soil and 15±1.5 nodules S. meliloti and S. medicae (data not shown). The ribotype /plant from the saline soil. Fifteen nodules per plant (15×5) diversity, estimated as the number of TRFs, was high and were used for isolation of bacteria according to similar in both soils, with 14.6±1.5 TRFs for the saline soil Vincent (1970). All 150 isolates were Gram negative, fast- and 16.3±3.8 TRFs for the agricultural soil. Comparing growing rhizobia, and formed single colonies with diameters agricultural and saline soil profiles, a total of 22 different of 2–3mmwithin2–3 days on yeast extract mannitol agar TRFs were found. Two TRFs were found exclusively on (YEM) plates. Isolates were then maintained on YEMA samples from each of the two different soils, one TRF medium slants at 4°C or stored in 20% glycerol at −80°C. (MspI at 84 bp) in the saline soil and one TRF (CfoIat 317 bp) in the agricultural soil. These two TRFs were Salt tolerance assigned by the MiCA3 (Shyu et al. 2007) to an uncultured alpha proteobacterium (MSB-4H3, accession number Bacterial isolates were incubated in liquid TY medium, DQ811855) and to an uncultured Mesorhizobium which contain 5 g/l tryptone, 3 g/l yeast extract, 10 mM (BME52, accession number DQ917826) respectively. An

CaCl2, with increasing concentrations of NaCl (0 mM, 100 mM, 300 mM, 600 mM and 1 M). In each tube, 200 μl from a 24-h pre-culture (OD620=1) was added to 5 ml fresh medium. Growth was recorded after 5 days of incubation at 150 rpm and 28°C by visual inspection. Petri dishes containing TY medium with the same NaCl concentrations were subdivided into squares, which were inoculated with 10 μl bacterial culture grown for 48 h in YEM broth (Elboutahiri et al. 2010). Salt tolerance was also estimated after 5 days of incubation at 28°C. Analysis was performed in triplicate for each isolate.

PCR-RFLP of 16S rDNA and 16S-23S rDNA intergenic spacer

Bacterial isolates from nodules were assigned to S. meliloti Fig. 1 Unweighted pair group method with arithmetic mean or S. medicae species according to the RsaI pattern of PCR- (UPGMA) dendrogram of the terminal restriction fragment length amplified 16S rRNA genes as previously described polymorphism (T-RFLP) profiles obtained after amplification of 16S rRNA genes of the Rhizobiaceae family from total DNA from (Laguerre et al. 1996; Biondi et al. 2003). Infra-specific agricultural (AS) and saline (SS) soils (1, 2, 3 are different replicate diversity of isolates was determined by PCR-RFLP analysis samples) 544 Ann Microbiol (2010) 60:541–547

Table 2 Salt tolerance of sino- a rhizobia isolates from Soliman Soil type Sinorhizobium species (no. of isolates) NaCl tolerance level soils 300 mM 600 mM 1 M

Saline soil S. medicae (17) 82.5% 17.5% 0 a Number of isolates growing at different NaCl concentrations and S. meliloti (58) 14% 72% 14% the percentage of isolates with Total (75) 28% 61% 11% respect to total isolates is reported Agricultural soil S. meliloti (75) 57% 43% 0 for each soil type

UPGMA dendrogram showing the relationships between agricultural than in the non-cultivated saline soil, in which the saline and the agricultural soil samples was then vegetation was more constant and several Medicago plants constructed (Fig. 1). All samples except AS2 clustered to grow spontaneously. their respective soil of origin, suggesting that members of Seventy-five rhizobial isolates for each soil type were the Rhizobiaceae are indeed differentiated in these two collected from nodules of M. truncatula, and 16S rRNA soils. This result confirms findings obtained in a previous gene sequences were analysed by PCR-RFLP. The 75 work analyzing the total bacterial communities in the same isolates trapped in the agricultural soil were all assigned to soils (Trabelsi et al. 2009). S. meliloti species as no isolates belonging to S. medicae were found in this soil (Table 2). On the contrary, in the Nodulating sinorhizobia populations saline soil, both sinorhizobia species were found. In particular, out of 75 isolates, 58 were identified as To assess the presence and titers of sinorhizobia nodulating belonging to S. meliloti and 17 to S. medicae. The presence Medicago species, MPN values were estimated using three of both S.meliloti and S. medicae has recently been Medicago species. Obtained titers varied from 1 x 102 to observed in marginal soils affected by salt and drought, in 3.3x104 g−1 soil (Table 1), in the range previously arid and semi-arid regions of Morocco (Elboutahiri et al. reported for the number of sinorhizobia in soil (Zribi et al. 2010), and a higher prevalence of S. meliloti over S. 2004). Interestingly, the sinorhizobial populations counted medicae is a common finding in agricultural and wild soils were globally higher by 1 Log unit in the saline clay soil (Talebi Bedaf et al. 2008). than in the non saline sandy one (one-way ANOVA, P< Analysing the intra-specific diversity of sinorhizobia 0.0001). Several factors, such as the presence of host isolates, PCR-RFLP of the 16S-23S rRNA IGS revealed the plants, clay and higher organic matter, may be related to presence of 19 different IGS types out the 150 isolates from the difference found (Øvreås and Torsvik 1998). Currently, both species (Fig. 2). The S. meliloti isolates accounted for the agricultural soil has been tilled once a year for several 14 IGS types, while 5 types were scored within S. medicae. years, and is used for co-cropping of olive and vegetables. IGS types G7, G11, G14 were the most represented (118/ Tillage could have determined a highly selective and more 150), comprising 87% of isolates from the agricultural soil homogenous environment (Saleena et al. 2002)inthe and 71% of isolates from the saline soil. The Shannon-

Fig. 2 Distribution of Sinorhi- zobium isolates among the 19 intergenic spacer types in the two soils Ann Microbiol (2010) 60:541–547 545

Table 3 Distribution of intergenic spacer types according to NaCl tolerance. IGS Intergenic spacer

Salt tolerance levela Species Number of isolates IGS type

300 mM S. meliloti 50 G6, G7, G8, G9, G10, G11, G12, G13 G14, G16, G17 S. medicae 14 G1, G2, G3, G4, G5 600 mM S. meliloti 75 G7, G11, G14, G18, G19 S. medicae 3G1 1M S. meliloti 8 G11, G15 S. medicae 0 a The ability to grow in different salt concentration was checked visually after 5 days of incubation in TY broth at 28°C

Wiener diversity indices (H’) for all the sinorhizobia it should be remembered that the life cycle of rhizobia and isolates (S. meliloti and S. medicae) were obviously higher the number of different micro-niches in soil hinder a simple in the saline (H’=1.96) than in the agricultural (H’=1.56) dose-response correlation for soil bacterial populations soil, given the presence of the two species in the former under very selective environments, probably allowing a soil. However, the opposite was found when only isolates large fraction of non-tolerant strains to survive and maintain belonging to S. meliloti were considered (H’=1.41 for good fitness also in highly selective environments. In the saline soil, 1.56 for agricultural soil). In fact, three IGS soils studied here, this could be especially relevant for S. types were present only in the saline soil, while ten were medicae, which showed lower levels of salt tolerance than identified within the agricultural soil isolates. S. meliloti but was recovered in salty soil only. The contribution of the host plant to the genetic diversity of No correlation between IGS types and salt-tolerance the nodulating rhizobia has already been demonstrated with phenotypes was found (Table 3). In fact, the same salt different leguminous species using molecular markers and tolerance phenotype was shared by more than one genotype symbiotic competence (Kuykendall et al. 1994;Bromfieldet and the same genotype contained isolates having different al. 1995). In the case of Medicago species, different host- salt-tolerance phenotypes. This result is in agreement with range species were observed reflecting a relative or absolute recent reports on rhizobia isolates from marginal soils of selectivity (Brockwell et al. 1988; Badri et al. 2003;Villegas Morocco (Elboutahiri et al. 2010) and from agricultural soil et al. 2006; Zribi et al. 2007). in Iran (Talebi Bedaf et al. 2008). The independence of salt- tolerant phenotypes and DNA fingerprinting genotypes was Salt tolerance noted also for the total culturable community of the same soils (Trabelsi et al. 2009); this evidence may indicate that For each of the 150 isolates, salt tolerance was evaluated in selection for salt tolerance could reside in transmissible both solid and liquid medium and showed identical results. genetic elements and/or that the appearance of salt tolerance Among the isolates, three main phenotypic classes were could be related to the mutation of one or few genes. In scored, with tolerance thresholds of 300 mM, 600 mM and fact, genetic determinants conferring high salt tolerance 1 M NaCl (Table 2). Interestingly S. meliloti isolates were were identified in systems such as Sinorhizobium fredii tolerant to higher NaCl concentrations than S. medicae. (Fujihara and Yoneyama 1994) and S. meliloti (Pocard et al. Comparison between the isolates of the two soils showed 1997; Talibart et al. 1997; Gouffi et al. 1999), suggesting that, as expected, a greater proportion of S. meliloti isolates that natural strains may harbor a wide plethora of genetic from the saline soil were tolerant to higher NaCl concen- systems able to cope with high salt concentrations in soil. trations compared with those from the agricultural soil. This Because of their high salt tolerance (1 M NaCl), the eight S. finding is in agreement with other reports indicating that meliloti isolates reported in this paper could represent bacteria isolated from saline environments are more likely preferred candidates for inoculant formulation in order to to survive under salt inhibitory concentrations than their promote cultivation of Medicago in saline soils. However, counterparts from non-saline habitats (Mpepereki et al. more work is still needed to confirm their usefulness in 1997; Elboutahiri et al. 2010). A number of studies have enhancing biological nitrogen fixation in environments shown that osmoadaptation in rhizobia appears to be affected by salinity or drought. atypical compared to that found in many enteric bacteria (Miller and Wood 1996), and rhizobia have been previously Acknowledgments This work was partially supported by the reported to colonize saline soil and thereby establish University of Firenze (Italy) “Scambi culturali e cooperazione effective symbiosis with plants (Zahran 1999). However, interuniversitaria internazionale (cap.f.s.2.16.04). 546 Ann Microbiol (2010) 60:541–547

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