The phenotypic, phylogenetic and symbiotic characterization of rhizobia nodulating Lotus sp. in Tunisian arid soils

Abstract

Fifteen bacterial isolates, representatives of different 16S rRNA-RFLP genomogroups which were isolated from root nodules of Lotus creticus and L. pusillus growing in the arid areas of Tunisia were characterized by phenotypic features and 16S rDNA sequences. Phenotypically, all isolates are fast growers with the ability to grow at a pH between 5.5 and 9. Most of the tested isolates tolerate NaCl concentrations from 1.39 to 3.48 %. Phylogenetically, the studied isolates are affiliated into the genera: Sinorhizobium (5 strains), Rhizobium (2 strains), and Mesorhizobium (4 strains). The 16S rDNA sequences of Tunisian Lotus sp. nodule isolates: LAC7511, LAC733, and Mesorhizobium alhagi (Alhagi sparsifolia symbiont) shared 100 % identical nucleotides similar to the 16S rDNA sequences of LAC831, LAC814 and Mesorhizobium temperatum CCNWSX0012-2 (Astragalus adsurgens symbiont). Non-nodulating bacteria, considered as endophytes of Lotus sp. nodules, were also found in our studies and they were classified into the genera: Phyllobacterium (2 strains), Starkeya (1 strain) and Pseudomonas (1 strain). Except for these four endophytic Lotus sp. bacteria, all other strains under investigation induce nodules on Lotus sp., but they differ in the number of induced root nodules and the effectiveness of atmospheric nitrogen fixation. The Sinorhizobium sp., Mesohizobium sp. and Lotus sp. nodule isolates, forming the most effective symbiosis with the plant host, are potential candidates for inoculants in revegetation programs.

Introduction

The plants belonging to the genera Lotus are now considered as prospective temperate legumes suitable for a wide application in Europe, Asia, Northern America and Africa. They are increasingly utilized in pastures throughout the world because of their high productivity over a wide range of soils (Blumenthal and McGraw 1999). They provide high quality animal fodder, prevent erosion and contribute to soil stabilization and ecosystem restoration (Fagg and Stewart 1994). The genus Lotus includes plants that are adapted to a wide range of habitats from marine environments to high altitudes, and from sandy to heavy saline soils (Heyn and Hernstradt 1967; Montes 1988). These crops have been promoted for low fertility pastures where they are more persistent and exhibit higher yields compared to traditional fodder legumes (Safronova et al. 2004).

Due to their capacity to enter into symbiosis with legume nodulating bacteria (LNB) collectively called rhizobia, Lotus sp. could play an important role in the nitrogen cycle. They may be used to restore or increase fertility of degraded and eroded soils. Studies of nodule bacteria isolated from indigenous legumes species growing in Tunisian arid zones showed their high diversity. They have been classified into the genera: Sinorhizobium, Rhizobium, Bradyrhizobium, Mesorhizobium, and Phyllobacterium (Zakhia et al. 2004, 2006; Mantelin et al. 2006; Rejili et al. 2009; Mahdhi et al. 2012). Rhizobia that nodulate Lotus species include both fast-growing Mesorhizobium loti (Jarvis et al. 1997) and slow-growing Bradyrhizobium sp. (Jordan 1982). At present, little information is available about the diversity and symbiotic properties of rhizobium strains nodulating Lotus sp. in Tunisia. Zakhia et al. (2004) found that strains isolated from Lotus creticus growing in the infra-arid region of Tunisia were the Rhizobium species. Recently, our research group in collaboration with a French group identified two new species called Ensifer numidicus sp. nov. and E. garamanticus sp. nov. which form symbiosis with Argyrolobium uniflorum and L. creticus (Merabet et al. 2010). Rejili et al. (2009) studied the genetic diversity of 60 isolates from the nodules of Lotus pusillus and L. creticus growing in arid environments in Tunisia by some molecular approaches including rep/PCR and PCR-RFLP of 16S rRNA gene analysis. Lotus sp. isolates are genomically very diverse. Some of them are affiliated to the genera Sinorhizobium, Rhizobium, and Mesorhizobium, but the taxonomic position of many other isolates has not been established. Therefore, the objective of this study is to determine the diversity of L. pusillus and L. creticus root nodule bacteria isolated by Rejili et al. (2009) and to clarify their taxonomic status by phenotypic and 16S rRNA gene sequence analyses, and also to determine their symbiotic effectiveness.

Materials and methods

Bacterial strains and cultural conditions

Fifteen bacteria, isolated by a standard method (Vincent 1970) from nodules of Lotus pusillus and L. creticus growing in the arid regions of Tunisia, were used in this study. They were chosen from among those previously characterized by RFLP analysis of 16S rDNA (Rejili et al. 2009). The isolates used in this study are shown in Table 1.

Table 1 Isolates used in this study, their hosts and their geographic origin

16S rRNA gene sequencing

Fifteen isolates representing each RFLP-16S rDNA genomogroups of 60 Lotus sp. microsymbionts described by Rejili et al. (2009) were cultured in YMA medium (Somasegaram and Hoben 1994). Bacterial DNA was prepared as described by Zakhia et al. (2004) or alternatively by the alkaline lysis method (Baele et al. 2000). The nearly full-length 16S rRNA gene was amplified using primers fD1 and rD1 (Weisburg et al. 1991) as described by Herrera-Cervera et al. (1999). PCR amplification was carried out in a 25-μl reaction mixture according to Rejili et al. (2009). The 16S rDNA amplicons were cleaned with Nucleofast filters. Two forward primers (FGPS6 and 16S-370f) and one reverse primer (FGPS1509) were used to obtain the complete and partial gene sequences. DNA was sequenced in the automated DNA sequencing service of Secugen (Madrid, Spain). DNA sequencing reactions were done in an Applied Biosystems PCR machine using the Big Dye 1.3 kit. After terminator elimination by CleanSeq from Agencourt, samples were electrophoresed in an AB3730 xl DNA sequencing machine. The 16S rDNA sequences of Lotus sp. symbionts and those of reference rhizobial species from GenBank were aligned by the Clustal X program and analyzed using Genedoc software package. A neighbor-joining tree was constructed and bootstrapped with 1,000 replications of each sequence using Mega software (Kumar et al. 2001). The GenBank accession numbers for the 16S rRNA gene sequences reported in this paper are JX962731 (LAC241), JX962732 (LAC231), JX962733 (LAC243), JX962734 (LAC513), JX962735 (LAC553), JX962736 (LAC733), JX962737(LAC742), JX962738 (LAC765), JX962739 (LAC813), JX962740 (LAC814), JX962741 (LAC831), JX962742 (LAC7511), JX962743 (LPS664), JX962744 (LPS715), and JX962745 (LPS811).

Phenotypic characterization

Twenty-six phenotypic features were used for characterization of studied isolates. The modified YMA medium (Somasegaram and Hoben 1994) was used to analyze the ability of isolates to use carbohydrates (1 % glucose and fructose) and amino acids (0.1 % L-arginine and L-leucine) as the sole carbon and nitrogen sources, respectively. The tolerance of isolates to pH was assessed by adjusting YMA medium pH to 5.5, 6.0, 7.0, 8.0, and 9.0 by the addition of sterile acid or alkali. The salt tolerance was analyzed by adding YMA medium NaCl to final concentrations of 1–4 %. Maximum growth temperature and antibiotic resistance (ampicillin 60 and 100 μg ml⁻¹, streptomycin 60 and 100 μg ml⁻¹, and kanamycin 60 and 100 μg ml⁻¹) were also assessed in YMA as described by Mohamed et al. (2000). Acid and alkali production was determined in YMA medium with bromothymol blue indicator (0.0025 %). In all experiments, growth was recorded after 72 h in triplicate.

Nodulation test and symbiotic efficiency

Symbiotic effectiveness of rhizobial strains was expressed in percent of dry weight of the aerial biomass of the plants inoculated with these bacteria to that of control uninoculated but fertilized plants, which were maintained with Jensen’s medium containing 0.1 M KNO₃. Seeds were surface-sterilized in 96 % sulfuric acid for 2 h, rinsed thoroughly with sterile distilled water, and germinated in Petri dishes at 20 °C. One seedling was transplanted into sterile plastic pots filled with autoclaved vermiculite. The pots were placed in a growth chamber at 23 °C with a 12- to 16-h photoperiod. Inoculation was performed with 10⁸–10⁹ cells of each isolate. The uninoculated plants (T_N: N-fertilized and uninoculated plants; and T₀: non-fertilized and uninoculated plants) were included as controls. Plants were harvested 10 weeks after planting and observed for nodulation. Shoots were cut off, dried at 70 °C for 48 h, then weighed. Nitrogen fixing effectiveness of nodules was determined by comparing the dry-shoot weight of inoculated plants with the dry-shoot weight of +N control plants.

Statistical analysis

Statistical analyses were performed with a SAS statistical package. Symbiotic properties were determined for each isolate in eight replications and using pair-wise comparison between the different treatments.

Results

16S rRNA gene sequencing

The nearly full-length sequences of 16S rRNA gene (1,375 bp) of 15 Lotus sp. nodule isolates were obtained by PCR reaction with primers fD1 and rD1 (Weisburg et al. 1991). The studied isolates are related to known LNB and are designated to the genera: Sinorhizobium, Mesorhizobium, Rhizobium, Phyllobacterium, Starkeya, and Pseudomonas. In the reconstructed phylogenetic tree (Fig. 1), five isolates (LAC243, LPS811, LAC765, LAC813 and LAC742) are located in the Sinorhizobium sp. branch. Strain LAC243 is closely related to Sinorhizobium meliloti Rm41. The 16S rDNA sequences of LPS811, LPS765, and Sinorhizobium meliloti BeTF2 are identical in 100 %. Strain LAC742 exhibited 99 % 16S rDNA sequence similarity with Sinorhizobium sp. CCBAU05593. Four strains, LAC7511, LAC733, LAC831. and LAC814. are grouped together with the Mesorhizobium species and all these strains formed on the phylogram common monophyletic cluster with a high bootstrap. The 16S rDNA sequences of LAC7511 and LAC733 strains as well as Mesorhizobium alhagi CCNWSX672 shared 100 % identical nucleotides, whereas 16S rDNA sequences of strains LAC831 and LAC814 were identical in 100 % with those of Mesorhizobium temperatum CCNWSX0012-2. LAC241 and LAC553 are closely related to Rhizobium huautlense RA16. The two Lotus sp. isolates, LPS715, LAC231, and Phyllobacterium sp. STM4021 indicated 100 % 16S rDNA sequence similarity. The 16S rDNA sequence analysis of LAC513 and LPS664 isolates revealed that these bacteria are phylogenetically related to Starkeya sp. ORS1474 and Pseudomonas sp. VTAE174, respectively.

Fig. 1

Phylogenetic analysis of Lotus creticus and Lotus pusillus nodule isolates based on the 16S rRNA gene sequences. Analyses were conducted using the Neighbor-Joining method, showing the relationships between isolates and reference rhizobial species. Bootstrap values >50 (using 100 replicates) are indicated at branching points. Bar 0.2 % estimated substitutions. GenBank accession numbers of the new isolates are shown in parentheses

Phenotypic analysis

Eleven Lotus sp. nodule isolates, identified by 16S rRNA gene sequence analysis as bacteria of the genera Mesorhizobium, Rhizobium, Sinorhizobium, and Phyllobacterium were examined for 26 phenotypic characteristics. Phenotypic features of studied strains are shown in Table 2. All tested strains were fast growers and acid producers on YMA medium with studied sugars as a sole carbon sources. They grew at 200, 300, 400, and 500 mM NaCl, except the LAC7511 and LAC733 strains (closely related to Mesorhizobium alhagi) which are able to grow at 400 and 500 mM NaCl. Two studied Sinorhizobium genus strains, LPS811 and LAC765, and two strains, LAC831 and LAC814, classified as Mesorhizobium temperatum exhibited the growth at 42 °C. All tested strains were able to grow at pH 5.5–9.0, except for LAC7511 and LAC733 (described by the analysis of 16S rDNA sequences as Mesorhizobium alhagi) which do not tolerate pH 5.5 and 6. Regarding sugar and amino acid utilization, the majority of the tested strains were able to use glucose–fructose and L-arginine–L-leucine as carbon and nitrogen sources, respectively. Exceptions were LPS715 and LAC231 isolates, classified as the genus Phyllobacterium strains, which are able to grow in the presence of fructose (as a sole carbon source) and L-arginine (as a sole nitrogen source). All strains were sensitive to streptomycin (100 μg ml⁻¹) and to kanamycin (60 and 100 μg ml⁻¹). Two strains, LPS715 and LAC231, identified by 16S rRNA gene sequences as belonging to the genus Phyllobacterium, LAC7511 and LAC733 strains closely related to Mesorhizobium alhagi, and LAC831 and LAC814 isolates classified as Mesorhizobium temperatum strains were resistant to 60 μg ml⁻¹of ampicillin and streptomycin. Lotus sp. LAC241 and LAC553 symbionts were only resistant to 60 μg ml⁻¹ of ampicillin. The tested strains classified as Sinorhizobium strains were diverse for antibiotic tolerance (Table 2).

Table 2 Results of phenotypic characteristics of Lotus sp. isolates (+ positive growth, − no growth)

Symbiotic properties

The studied Lotus sp. nodule isolates, identified by 16S rRNA gene sequence analysis as the members of the genera: Mesorhizobium, Rhizobium, Sinorhizobium (=Ensifer), Phyllobacterium, Starkeya, and Pseudomonas were evaluated for their symbiotic potential on their original hosts (Table 3). All isolates, except LAC513, LAC231, LPS715, and LPS664, were able to form symbiosis with plants from which they were isolated. Studied Lotus sp. symbionts induced root nodules globular in shape with a pink-red pigmentation on their plant hosts. Two isolates, LAC733 (classified to the genus Mesorhizobium) and LAC813 (designated as member of the genus Sinorhizobium = Ensifer), gave significantly higher nodule numbers per Lotus sp. plant than the others (P  < 0.05). Analysis of dry biomass of plant aerial part, used as an indicator of relative N₂ fixation effectiveness, indicated that strain LAC765 (Sinorhizobium = Ensifer) was the most effective diazotroph with dry weight biomass of 91.46 ± 0.01 in respect to the T_N control (P  < 0.05). The least effective isolate in nitrogen fixation was the LAC742 strain with only 62.41 ± 0.05 of the dry biomass with respect to the T_N control. The results of symbiotic performance of studied LAC765, LAC814, LAC243, and LAC7511strains in laboratory plant tests showed that these four isolates enter into effective mutualistic interactions with Lotus sp. (relative effectiveness ≥70 %).

Table 3 Results of symbiotic potential of root wild legume nodulating bacteria

Discussion

Knowledge on genetic and functional diversity of root-nodule bacteria associated with the indigenous legume flora is very useful for inoculant strain selection. In this study, 15 bacterial isolates, representing different 16S rRNA-RFLP genomogroups, which were isolated from root nodules of Lotus creticus and L. pusillus and studied earlier by Rejili et al. (2009) were characterized by phenotypic features and 16S rDNA sequence analysis.

By using the comparative 16S rRNA gene sequence analysis, Lotus sp. isolates are grouped on the phylogram in the Sinorhizobium and Rhizobium genera cluster, as are many other indigenous legume symbionts from Tunisia (Zakhia et al. 2004; Ben Romdhane et al. 2006; Mahdhi et al. 2012), and also located in the Mesorhizobium species branch as described for the first time by Rejili et al. (2009) in the case of Lotus sp. nodule isolates derived from Tunisian arid soil. Among our Lotus sp. isolates, Bradyrhizobium genus strains were not detected; however, Jordan (1982) reported that such slow-growing rhizobia are Lotus species symbionts. Similarly to our results, León-Barrios and Donate-Correa (2007) found that rhizobia nodulating Lotus sp. derived from Spain belong to the genera: Mesorhizobium, Sinorhizobium, and Rhizobium. In our studies, we showed that 16S rRNA gene sequences of strains LAC7511 and LAC733 as well as Mesorhizobium alhagi CCNWSX672 (symbiont of Alhagi sparsifolia; Chen et al. 2010) shared 100 % identical nucleotides. The 16S rRNA gene sequences of the two LAC831 and LAC814 strains indicate high similarity rates to 16S rDNA sequence of Mesorhizobium temperatum CCNWSX0012-2, (nodulating Astragalus adsurgens; Gao et al. 2004). Close phylogenetic relationships between bacteria nodulating Lotus edulis and L. ornithopodioides as well as L. cytisoides and Mesorhizobium loti type strain were documented by Safronova et al. (2004) on the basis of 16S rDNA PCR-RFLP analysis. Among Lotus tenuis symbionts isolated in Argentina by Estrella et al. (2009) some of them were closely related to M. amorphae, M. mediterraneum, M. tianshanense, and the type strain M. loti NZP2213.

In our collection of Lotus sp. nodule isolates, studied by 16S rRNA gene sequence analysis, two strains, LPS715 and LAC231, are classified as Phyllobacterium and two others, i.e. LPS664 and LAC513, are identified as the members of the genus Pseudomonas and Starkeya, respectively. The 16S rDNA sequences of two isolated Phyllobacterium strains are similar to the 16S rRNA gene sequence of Phyllobacterium sp. STM4021, which was isolated by Mahdhi et al. (2007) from the root nodules of Genista saharae growing in arid Tunisian soil. Legume endophytes have also been reported among the genus Pseudomonas bacteria (Elvira-Recuenco and Van Vuurde 2000). We also isolated the genus Pseudomonas strains from the nodules of Hedysarum spinosissimum (Mahdhi et al. 2012), similar to Benhizia et al. (2004) and Muresu et al. (2008). Shiraishi et al. (2010) reported that Pseudomonas genus strains formed root nodules on black locust with differentiated nodule tissues. Zakhia et al. (2006) found that one strain isolated from root nodules of Lotus argenteus, growing in the infra-arid regions of Tunisia, belongs to the genus Starkeya, but it failed to nodulate its original plant host in vitro. The genera studied by us the, Phyllobacterium, Pseudomonas, and Starkeya strains, failed to form nodules on Lotus creticus and L. pusillus in laboratory plant tests, although they were isolated from these legumes. We suppose that these bacteria entered nodules due to co-infection with natural Lotus sp. symbionts. Further investigations are needed to explain this phenomenon.

Phenotypically, all tested isolates were acid producers, sensitive to 100 μg ml⁻¹ of ampicillin and kanamycin, and able to grow at pH 5.5–9.0, except LAC7511 and LAC733 strains (described as closely related to Mesorhizobium alhagi) which do not tolerate pH 5.5 and 6. Cooper (1982) reported that rhizobial strains which nodulate Lotus sp. show marked differences in acid tolerance. Among Lotus sp. symbionts are moderately slow-growing Mesorhizobium loti (Jarvis et al. 1997), tolerant to pH 4.5, and slow-growing Bradyrhizobium sp. (Jordan 1982) which are sensitive to this pH. As for salinity tolerance, our results showed that only two isolates, i.e. LAC7511 and LAC733, are enable to grow at 400 and 500 mM NaCl. Safronova et al. (2004) described rhizobia nodulating L. edulis, L. ornithopodioides, and L. cytisoides which tolerate up to 2 % NaCl. Lotus sp. nodule bacteriaI isolated by us and identified as members of the genus Sinorhizobium, were even able to grow at 35°–40 °C, and two of them also multiply at 42 °C (Table 2). Safronova et al. (2004) reported that Lotus sp. mesorhizobium symbionts cannot grow at temperatures higher than 35 °C. It has been reported that many rhizobia form symbiosis with legumes in Tunisia (Zakhia et al. 2004; Zribi et al. 2004; Rejili et al. 2009; Mahdhi et al. 2012) and most new Lotus sp. nodule isolates described in this paper tolerate a high temperature (40 °C) as well as high NaCl concentration (up to 3 %). This may be an effect of specific adaptation to the high temperatures and soil salinity that characterize arid regions (Karanja and Wood 1988; Abdelmoumen et al. 1999).

Lotus sp. LAC765 symbiont from our collection, closely related to Sinorhizobium genus bacteria, is the most efficient in nitrogen fixation strain among studied isolates (Table 3). Its ability to grow at high temperature (42 °C) and high NaCl concentration (500 mM) renders this LAC765 isolate as a promising bacterium for Lotus sp. inoculation, provided that this strain possess high competitiveness for nodule occupancy.

In conclusion, our investigation showed that LNB originating from root nodules of Lotus creticus and L. pusillus growing in arid Tunisian soil are genetically diverse and they are designated to the genera: Sinorhizobium, Rhizobium, and Mesorhizobium. Among Lotus sp. nodule isolates were bacteria which in laboratory plant tests did not show the ability to induce these root structures. They were classified into the genera Phyllobacterium, Starkeya, and Pseudomonas. In order to provide further information on the diversity of rhizobia forming symbiosis with Lotus sp. growing in Tunisia, further studies are necessary and such investigations are in progress.