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Medicago ciliaris growing in Tunisian soils is preferentially nodulated by Sinorhizobium medicae.


Many species of the legume genus Medicago are native to the Mediterranean basin (Lesins and Lesins 1979) and are important as agricultural crops (Irwin et al. 2001). Compared to M. sativa, the most important species for cultivation, and M. truncatula, the model chosen for studies in nitrogen fixation (Cook 1999; de Billy et al. 2001; Ben Amor et al. 2003), investigations with M. ciliaris as the focus have been very limited (Laouar and Abdelguerfi 2000). The Medicago species Medicago ciliaris is an annual that is tolerant to salt stress (Abdelly et al. 1995) and may show promise for cultivation in salt-affected soils. Because this species grows in soils that are heavy with clay it has application as a cover crop, in pastures, or for producing forage (Laouar and Abdelguerfi 2000). The natural habitat of M. ciliaris is in the humid and sub-humid regions of north Tunisia where it is an important pasture.

Although most Medicago species form symbioses with the 2 species Sinorhizobium meliloti and S. medicae (Brunel et al. 1996; Rome et al. 1996), it is becoming evident that several different species of Medicago may have dissimilar affinities for infection by these 2 rhizobial species. For example, Garau et al. (2005) demonstrated that S. medicae frequently nodulated Medicago species that are adapted to acid soils, while S. meliloti formed symbioses with those growing in more alkaline to neutral soils. Bena et al. (2005) indicated that the geographic distribution of these rhizobial species appeared related to the incidence of the species of Medicago resulting from the characteristics of the soils. From initial studies it would appear that M. ciliaris growing in Tunisian soils is nodulated by both rhizobial species (Jebara et al. 2001), even though S. meliloti was reported to form inefficient symbioses with this host legume species (Bena et al. 2005).

The diversity of rhizobia-nodulating Medicago has been widely reported (Brunel et al. 1996; Rome et al. 1996; Paffetti et al. 1998; Carelli et al. 2000; Roumiantseva et al. 2002; Bailly et al. 2006; van Berkum et al. 2006). The majority of these reports were concentrated on an analysis of M. sativa and S. meliloti (Bromfield et al. 1995; Paffetti et al. 1996; Hartmann et al. 1998; Andronov et al. 1999), while information, on the rhizobia-nodulating M. truncatula (Zribi et al. 2004, 2005) and M. laciniata (Villegas et al. 2006) was more recently reported. It is clear from these reports that there are molecular differences between the 2 species of Sinorhizobium that may be useful for subsequent investigations of rhizobia that nodulate other Medicago species.

Because M. ciliaris is an important legume of northern Tunisia that has received relatively little attention, its interaction with rhizobia was further elucidated. The objectives of this work were to: (i) survey nodulation and growth of M. ciliaris grown in Tunisian soils, (ii) analyse the diversity of rhizobia that nodulated this plant, and (iii) examine the symbiotic performances of M. ciliaris with several of the isolates that were obtained.

Materials and methods

Cultures and nodule sampling

Soil sampling and pods of M. ciliaris were collected from 4 different geographic regions in Tunisia. The pods were allocated to 4 different populations of Medicago ciliaris: TNC1 (Enfidha, centre of Tunisia), TNC8 (Soliman, Noah of Tunisia), TNC10 (Rhayet, Noah of Tunisia), and TNC11 (Mateur, North of Tunisia), based on morphological and molecular markers (Y. Badri et al., unpublished data). In addition to these 4 regions of Tunisia, one more soil sample was collected from Jelma in southern Tunisia. Bioclimatic conditions and abundance of different Medicago species at the sampling sites have been summarised by Zribi et al. (2004) with the exception of Mateur, which is located in the sub-humid area.

Seeds were sterilised and scarified according to Vincent (1970). The treated seeds were sown in the corresponding soil samples from each location. One plant was grown in each soil contained in sterilised plastic pots that were placed in a growth chamber at 25 [degrees] C with 16-h photoperiod and 80% relative humidity. Isolations were made using 3 replicate plants grown in each of the 5 soils. Nodules were collected when the plants were 60 days old and rhizobia were isolated (1 isolate per nodule) and purified according to Vincent (1970). The number of nodules in each of the treatments were determined and the plant shoots were dried at 70 [degrees] C to determine their dry weights.

Molecular characterisation of the isolates

The DNA of 100 isolates (20 isolates from each location) was extracted and was used in PCR to amplify the 16S rRNA gene as described by Zribi et al. (2004). The 16S rRNA gene PCR products were digested with RsaI and the restriction fragments were separated by horizontal agarose gel electrophoresis to distinguish S. meliloti and S. medicae (Laguerre et al. 1997; Biondi et al. 2003; Zribi et al. 2004) among the isolates. Twenty isolates that were assigned to S. medicae based on the 16S rRNA analysis and that were isolated from soils of the first 4 locations were randomly chosen for PCR/RFLP analyses of the intergenic region between nifD and nifK (Jamann et al. 1993) and of the nodC gene (Laguerre et al. 2001) using MspI digests.

Genetic diversity among 38 randomly chosen isolates of S. medicae that originated from the first 4 locations was determined by using REP-PCR (De Bruijn 1992). The fingerprint patterns resulting from horizontal gel electrophoresis of the PCR products were used to obtain a binary matrix (0/1), where the 0 and the 1 represented the absence or the presence of a PCR product of a specific molecular size, respectively. The binary matrix was used to obtain a dendrogram using the Unweighted Pair Group with Mathematical Average (UPGMA) algorithm (STATISTICA software, France) that portrayed the similarities among the isolates.

Competitiveness and symbiotic performances of isolates on M. ciliaris grown on nutrient media

Seeds of M. ciliaris line TNC10-11 were surface-sterilised and scarified and were then placed on the surface of 30-mL nutrient media (Fahraeus 1957) slants in 2 by 20cm tubes that were placed in a growth chamber as described above. The seedlings were inoculated 48 h later with 300 [micro]L YEM-grown, early stationary phase ([10.sup.8] bacteria/mL), broth suspensions. The inocula were prepared as 100-mL YEM broth cultures that were shaken at 150 r.p.m. on a rotary shaker for 48 h. The isolates of S. medicae used were chosen according to their placement on the dendrogram derived from the REP-PCR analysis and subsequent preliminary plant tests. The 4 isolates of S. medicae E8 (from Enfidha), and M1, M3, and M6 (from Mateur), and the 1 isolate or S. meliloti J2 (from Jelma) were tested separately with M. ciliaris. In addition, the 2 isolates E8 and J2, representing the 2 species S. medicae and S. meliloti, respectively, were used in equal numbers to co-inoculate M. ciliaris in a competition for nodulation analysis.

Non-inoculated plants were used as controls and each treatment was tested with 10 replicates of culture. The number of nodules and the nitrogen fixation efficiency, established as the ratio of the shoot dry matter of inoculated plants and non-inoculated plants, were determined for each treatment after 2 months. From nodules of co-inoculated plants rhizobia were isolated and characterised by 16S rRNA PCR-RFLP. The percentage of nodules occupied by S. medicae of each plant was calculated. Duncan's Multiple Range tests at P = 0.05 were used to identify significant differences among the treatments.


Growth and nodulation of M. ciliaris in soils from the four regions of Tunisia

M. ciliaris grown in soil of the Mateur region was significantly more nodulated than when grown in soils of the other 3 regions, and the smallest number of nodules was observed on plants grown in soil of the Soliman region (Table 1). Very poor nodulation of M. ciliaris collected from the Soliman region was observed when grown in soil from Jelma, where this Medicago species is naturally absent. Generally, there was less variation in plant growth than nodulation, since only the shoot dry matter of plants grown in Soliman soil was significantly lower than those grown in soils from the other 3 regions (Table 1). The shoot dry matter of M. ciliaris grown in Jelma soil was the lowest, which was probably because of the poor nodulation.

Genetic analysis of the isolates

All but one of the isolates obtained from the soils of Enfidha, Mateur, Rhayet, and Soliman using M. ciliaris as trap host were identified as S. medicae based on RsaI digests of the PCR-generated 16S rRNA gene. The single exception was an isolate from Enfidha, which had a 16S rRNA fingerprint identified with S. meliloti. Based on this analysis, all the 20 isolates from nodules of M. ciliaris grown in soil of Jelma were identified with S. meliloti. From this result, it was concluded that M. ciliaris could be nodulated by S. meliloti if S. medicae is absent as is the case with soil of Jelma (Zribi et al. 2004).

Diversity of the isolates identified as S. medicae

From the electrophoretic separation of REP-PCR products, 31 different fingerprint patterns were distinguished among 38 isolates identified with S. medicae based on 16S rRNA analyses. Three of the PCR products that were of approximate molecular sizes of 2200, 1700, and 600 bp were common to each lane. Most of the isolates were very similar except for isolates E2 and S4 (Fig. 1). A relationship of REP-PCR pattern with host or with soil of origin was not apparent. PCR-RFLP patterns of the nifDK and the nodC analysis were monomorphic across the 38 isolates (data not shown).


Infectivity and symbiotic performance of several isolates of S. medicae and S. meliloti with M. ciliaris

Medicago ciliaris line TNC10-11 originating from Rhayet inoculated with S. meliloti isolate J2 grew poorly and at the time of harvest had fewer leaves than plants inoculated with several of the other isolates that were placed with S. medicae by 16S rRNA PCR-RFLP. Nodules produced with isolate J2 typically were ineffective, while inoculation with the other 4 isolates led to nodules that were characteristically effective (Table 2). Significant variation in nodule number but not in nitrogen fixation efficiency among the isolates identified as S. medicae was detected (Table 2).

A co-inoculation experiment with S. medicae isolate E8 and S. meliloti isolate J2 was used to provide further evidence that nodulation of M. ciliaris by S. medicae was favoured over S. meliloti when both are present in the root-zone. From characterisation of the isolates originating from all the nodules, it was shown that 80% were occupied by isolate E8. The isolate J2 was recovered from 20% of the nodules, which had typically ineffective morphologies (Table 2).


With this investigation, evidence has been provided that nodulation of M. ciliaris is variable in soils of the different climatic zones of Tunisia where this legume species is native. With Medicago such variability in nodulation across different soils of the native habitats in Tunisia has not been reported before. However, potentially this phenomenon was a possibility since variability in nodulation of chickpea was reported across 19 sites of Tunisia (Aouani et al. 2001). Although the reasons for site-dependent variability in nodulation of these 2 legumes across Tunisia are not clear, it is possible that dissimilarities in the rhizobia and/or the soils at each site may have had an influence on the establishment of root nodules (Mendes and Bottomley 1998; Zahran 1999).

A more extreme example of the variability in nodulation of M. ciliaris that was encountered was in soils of the Jelma region, where plant growth was poor and nodulation was sparse. In this case, the observation can be explained by the rhizobia present in the soil and their potential interaction with M. eiliaris. Soil of the Jelma region was reported to be colonised by Medicago-nodulating rhizobia that had characteristics of S. meliloti, while those of S. medieae were absent (Zribi et al. 2004). This would explain the reason why the nodules of M. ciliaris grown in Jelma soil only yielded isolates that were subsequently characterised as S. meliloti. Also, Bena et al. (2005) have indicated that the symbiotic interaction of S. meliloti and M. ciliaris is inefficient, which was confirmed in this investigation. Therefore, the presence of only S. meliloti in Jelma region soil and their ineffective nodulation of M. ciliaris would explain the poor plant growth. It is therefore possible that M. ciliaris is not part of the native flora in the Jelma region because this legume species has not been able to compete with other Medicago species that do form effective symbioses with S. meliloti.

In contrast to the Jelma region, evidence was provided in this investigation that soils at the other 4 sites do contain Medicago-nodulating rhizobia that share characteristics with S. medicae. This observation is supported by Zribi et al. (2004), who reported that 3 of these sites yielded S. medicae when isolations were made using M. truncatula as trap host. The superior plant growth at the other 4 sites where S. medicae is indigenous, compared to the Jelma region where S. medicae is absent, can be explained because S. medicae reportedly does form effective symbioses with M. ciliaris (Bena et al. 2005).

Zribi et al. (2004) reported the presence of S. meliloti in soils of the Rhayet and Enfidha regions, which were used in this study to recover rhizobia with M. ciliaris. Even though S. meliloti is present in these 2 soils, none were recovered. Instead, all nodules of M. ciliaris grown in these soils yielded rhizobia that were shown to share characteristics with S. medicae. Possibly this observation could be explained by host influence on the competition for nodulation by these 2 different rhizobial species. Further evidence for host influence on nodulation was obtained in a competition for nodulation experiment in test tubes where co-inoculation of an isolate each of S. meliloti and S. medicae led to a significant disproportion of nodules occupied by the latter. Host influence on nodule occupancy is not confined to M. ciliaris, since M. polymorpha and M. laciniata were shown to preferentially nodulate with S. medicae (Brunel et al. 1996) and with S. meliloti (Badri et al. 2003; Villegas et al. 2006), respectively. Although specific genetic determinants involved with infection could be responsible for the host's influence on nodule occupancy as with the nodC allele and M. laciniata (Barran et al. 2002), reasons for other ineffective combinations are unclear.


The authors thank M. Badri for providing seeds of Medicago ciliaris and A. Abdelguerfi and M. Laouar for helpful discussion.

Manuscript received 28 February 2007, accepted 28 August 2007


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K. Zribi (A,C), Y. Badri (A), S. Saidi (A), P. van Berkum (B), and M. E. Aouani (A)

(A) Laboratoire Interactions Legumineuses Microorganismes (LILM), Centre de Biotechnologie, Technopole de Borj Cedria, BP901, Hammam lif 2050, Tunis, Tunisie.

(B) SGIL, Bldg-006, BARC-West, ARS, U.S. Department of Agriculture, 10300 Baltimore Blvd, Beltsville, MD 20705, USA.

(C) Corresponding author. Email:
Table 1. Nodulation and growth of M. ciliaris
grown indifferent Tunisian soils

Within a column, differences among means of 12 plants followed by
a different letter are significant at P=0.05 by Duncan's multiple
range test. SDM, Shoot dry matter

Populations of Soil of Texture Bio climatic stage
M. ciliaris culture

Enfidha Enfidha Silt-sandy Inferior semi-arid
Mateur Mateur Silt-clayey Sub-humid
Rhayet Rhayet Fine silt Sub-humid
Soliman Soliman Fine silt Superior semi-arid
Soliman Jelma Silt-sandy Superior arid

Populations of Nodules SDM
M. ciliaris per plant (g/plant)

Enfidha 50.7 b 0.57 a
Mateur 73.4 a 0.51 a
Rhayet 46 b 0.60 a
Soliman 25.5 c 0.32 b
Soliman 2.3 d 0.11 c

Table 2. Nodulation and nitrogen fixation efficiency (NFE) of M.
ciliaris originating from Rhayet and inoculated separately with
isolates of S. medicae and of S. meliloti or co-inoculated with
one isolate of each

The line TNC10-11 of M. ciliaris grown on the surface of 30mL nutrient
media (Fahraeus 1957) slants in 2 by 20 cm tubes using 10 replicates
for each treatment. Within a column, differences among means followed
by a different letter are significant at P=0.05 by Duncan's multiple
range test

 Geographic Nodules
Strains Species origin per plant NFE

E8 S. medicae Enfidha 12.8 a 1.58 a
J2 S. meliloti Jelma 3.3 b 0.65 e
M1 S. medicae Mateur 10.3 a 1.43 ab
M6 S. medicae Mateur 11 a 1.11 bcd
M3 S. medicae Mateur 3.7 b 1.24 abc
E8 and J2 -- -- 3.4 b 1.05 cd

 Morphology of Nodular
Strains nodules occupancy (%)

E8 Longed, foiled and pink 100
J2 Spherical, white and rudimentary 100
M1 Longed, foiled and pink 100
M6 Longed, foiled and pink 100
M3 Longed, foiled and pink 100
E8 and J2 The two types 80.9 (E8) and
 19.1 (J2)
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Author:Zribi, K.; Badri, Y.; Saidi, S.; van Berkum, P.; Aouani, M.E.
Publication:Australian Journal of Soil Research
Article Type:Report
Geographic Code:6TUNI
Date:Sep 1, 2007
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