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RESEARCH ARTICLE

Genetic characterisation and symbiotic proprieties of native sinorhizobia trapped by Medicago sativa on Tunisian soils

S. Saidi A , K. Zribi A C , Y. Badri A and M. E. Aouani B
+ Author Affiliations
- Author Affiliations

A Laboratoire Interactions Légumineuses Microorganismes (LILM), Centre de Biotechnologie, Technopole de Borj Cedria, BP 901, Hammam lif 2050, Tunis, Tunisie.

B NEPAD/North Africa Biosciences Network, National Research Center, El Buhouth St, Cairo 12311, Egypt.

C Corresponding author. Email: kais.zribi@cbbc.rnrt.tn

Australian Journal of Soil Research 47(3) 321-327 https://doi.org/10.1071/SR08014
Submitted: 16 January 2008  Accepted: 17 November 2008   Published: 25 May 2009

Abstract

Medicago sativa, a perennial species of the genus Medicago, is particularly cultivated in the Tunisian oases. Three M. sativa cultivated varieties (Magali A, Gabès2355, ABT805) and 1 genotype from a local provenance (Widhref) were cultivated on 4 Tunisian soil samples exhibiting contrasting pedo-climatic characteristics. Results showed that nodulation and aerial growth were significantly dependent on both variety and soil factors. Magali A showed the highest number of nodules, and varieties grown in Amra and Mateur soils produced the largest nodulation and aerial growth.

A collection of 250 rhizobial isolates was obtained from the 4 soils by trapping them on M. sativa. Molecular characterisation by PCR/RFLP of 16S genes showed that almost all isolates (158 of 160 isolates) belong to Sinorhizobium meliloti. The subsequent analysis of isolates from 2 contrasting soils (Mateur, in the north of Tunisia, and Rgim Maatoug, in the south) demonstrated that strains harbour polymorphism at symbiotic genes, with 4 nodC genotypes, 2 nifDK genotypes and 2 nodA genotypes. The 2 soils contained different symbiont genotypes. The distribution of various profiles types was different according to geographic sites. The soils of Mateur and Rgim Maatoug do not contain the same genotypes. Eleven representative strains from this collection were evaluated for their nodulation and nitrogen fixation capacities. Results showed a high degree of variability among strains for nodulation and aerial growth parameters, and only 2 strains, GII/M1 and LII/M1, seem to be effective with all 4 varieties of M. sativa tested. Strains isolated from the soil of northern Tunisia were more efficient and produced fewer nodules than those trapped on southern soil.

Additional keywords: M. sativa, PCR/RFLP, S. meliloti, molecular polymorphism, symbiotic genes, soil, efficient strains.


Acknowledgments

Financial support of this research was provided by the Project FP6 PERMED PL 509140: ‘Improvement of native perennial forage plants for sustainability of Mediterranean farming systems’. The authors would also like to thank Mr. Badri M. for the helpful discussion, Mr. Casa E. for the beneficial comments and Ms. Friesen M. for English editing.


References


Andronov EE, Roumyantseva ML, Sagoulenko VV, Simarov BV (1999) Effect of the host plant on the genetic diversity of a natural population of Sinorhizobium meliloti. Russian Journal of Genetics 35, 1169–1176.
CAS |
open url image1

Badri Y, Zribi K, Badri M, Huguet T, Aouani ME (2003) Sinorhizobium meliloti nodulates Medicago laciniata in Tunisian soils. Journal of Genetics and Plant Breeding 39, 178–183. open url image1

Bailly X, Olivieri I, De Mita S, Cleyet-Marel J, Bena G (2006) Recombination and selection shape the molecular diversity pattern of nitrogen-fixing Sinorhizobium sp. associated to Medicago. Molecular Ecology 15, 2719–2734.
CAS | PubMed |
open url image1

Béna G (2001) Molecular phylogeny supports the morphologically based taxonomic transfer of the “medicagoid” Trigonella species to the genus Medicago L. Plant Systematics and Evolution 229, 217–236.
Crossref | GoogleScholarGoogle Scholar | open url image1

Béna G, Lyet A, Huguet T, Olivieri I (2005) MedicagoSinorhizobium symbiotic specificity evolution and the geographic expansion of Medicago. Journal of Evolutionary Biology 18, 1547–1558.
PubMed |
open url image1

Béna G, Prospéri JM, Lejeune B, Olivieri I (1998) Evolution of annual species of the genus Medicago: A molecular phylogenetic approach. Journal of Molecular Evolution 9(3), 552–559. open url image1

Biondi EG, Pilli E, Giuntini E, Roumiantseva ML, Andronov EE , et al. (2003) Genetic relationship of Sinorhizobium meliloti and Sinorhizobium medicae strains isolated from Caucasian region. FEMS Microbiology 220, 207–213.
Crossref | GoogleScholarGoogle Scholar | CAS | open url image1

Bradić M, Sikora S, Redžepović S, Štafa Z (2003) Genetic Identification and symbiotic efficiency of an indigenous Sinorhizobium meliloti field population. Food Technology and Biotechnology 41(1), 69–75. open url image1

Brockwell J, Bottomley PJ (1995) Recent advances in inoculant technology and prospects for the future. Soil Biology & Biochemistry 27, 683–697.
Crossref | GoogleScholarGoogle Scholar | CAS | open url image1

Bromfield ESP, Sinha IB, Wolyentz MS (1986) Influence of location, host cultivar and inoculation in the composition of naturalized populations of Rhizobium meliloti in Medicago sativa nodules. Applied and Environmental Microbiology 51, 1077–1084.
PubMed |
open url image1

Carelli M, Gnocchi S, Fancelli S, Mengoli A, Paffetti D, Scotti C, Bazzicalupo M (2000) Genetic diversity and dynamics of Sinorhizobium meliloti populations nodulating differents alfalfa cultivars in italian soils. Applied and Environmental Microbiology 66(11), 4785–4789.
Crossref | GoogleScholarGoogle Scholar | CAS | PubMed | open url image1

Downie JA (1998) ‘Functions of rhizobial nodulation genes.’ In ‘The Rhizobiaceae’. (Eds HP Spaink, A Kondorosi, PJJ Hooykaas) pp. 387–402. (Kluwer Academic Publishers: Dordrecht, Netherlands)

Eardly BD, Materon LA, Smith NH, Johnson DA, Rumbaugh MD, Selander RK (1990) Genetic structure of natural populations of the nitrogen-fixing bacterium Rhizobium meliloti. Applied and Environmental Microbiology 56, 187–194.
CAS | PubMed |
open url image1

Evans PM, Howieson JG, Nutt BJ (2005) Increased yield and persistence of several annual medic species and Medicago sativa by inoculation with selected strains of Sinorhizobium. Australian Journal of Experimental Agriculture 45, 217–224.
Crossref | GoogleScholarGoogle Scholar | open url image1

Fãhraeus G (1957) The infection of clover root hairs by nodule bacteria studied by a simple glass slide technique. Journal of General Microbiology 16, 374–381.
PubMed |
open url image1

Garau G, Reeve WG, Brau L, Deiana P, Yates RJ, James D, Tiwari R, O’Hara GW, Howieson JG (2005) The symbiotic requirements of different Medicago spp. suggest the evolution of Sinorhizobium meliloti and S. medicae with hosts differentially adapted to soil pH. Plant and Soil 276, 263–277.
Crossref | GoogleScholarGoogle Scholar | CAS | open url image1

Heath KD, Tiffin P (2007) Context dependence in the coevolution of plant and rhizobial mutualists. Proceedings of the Royal Society of London. Series B. Biological Sciences 274(1620), 1905–1912.
Crossref | GoogleScholarGoogle Scholar | open url image1

Jamann S, Fernandez MP, Normand P (1993) Typing method for N2-fixing bacteria based on PCR/RFLP—application to the characterisation of Frankia strains. Molecular Ecology 2, 17–26.
Crossref | GoogleScholarGoogle Scholar | CAS | PubMed | open url image1

Jebara M, Mhamdi R, Aouani ME, Ghrir R, Mars M (2001) Genetic diversity of Sinorhizobium populations recovered from different Medicago varieties cultivated in Tunisian soils. Canadian Journal of Microbiology 47, 139–147.
Crossref | GoogleScholarGoogle Scholar | CAS | PubMed | open url image1

Laguerre G, Nour SM, Macheret V, Sanjuan J, Drouin P, Amarger N (2001) Classification of rhizobia based on nodC and nifH gene analysis reveals a close phylogenetic relationship among Phaseolus vulgaris symbionts. Microbiology 147, 981–993.
CAS | PubMed |
open url image1

Laguerre G, van Berkum P, Amarger N, Prevost D (1997) Genetic diversity of rhizobial symbionts isolated from legume species within the genera Astragalus, Oxutropis, and Onobrychis. Applied and Environmental Microbiology 63, 4748–4758.
CAS | PubMed |
open url image1

Mehmannavaz R, Prasher S, Ahmed D (2002) Rhizospheric effects of alfalfa on biotransformation of polychlorinated biphenyls in a contaminated soil augmented with Sinorhizobium meliloti. Process Biochemistry 37, 955–963.
Crossref | GoogleScholarGoogle Scholar | CAS | open url image1

Mhadhbi H, Jebara M, Limam F, Huguet T, Aouani ME (2005) Interaction between Medicago truncatula lines and Sinorhizobium meliloti strains for symbiotic efficiency and nodule antioxidant activities. Physiologia Plantarum 124, 4–11.
Crossref | GoogleScholarGoogle Scholar | CAS | open url image1

Mhamdi R, Laguerre G, Aouani ME, Mars M, Amarger N (2002) Different species and symbiotic genotypes of field rhizobia can nodulate Phaseolus vulgaris in Tunisian soils. FEMS Microbiology Ecology 41, 77–84.
Crossref | GoogleScholarGoogle Scholar | CAS | open url image1

Mnasri B, Mrabet M, Laguerre G, Aouani ME, Mhamdi R (2006) Salt tolerant rhizobia isolated from a Tunisian oasis that are highly effective for symbiotic N2-fixation with Phaseolus vulgaris constitute a novel biovar (bv. Mediterranense) of Sinorhizobium meliloti. Archives of Microbiology 187(1), 79–85.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Paffetti D, Daguin F, Fancelli S, Gnocchi S, Lippi F, Scotti C, Bazzicalupo M (1998) Influence of plant genotype on the selection of nodulating Sinorhizobium meliloti by Medicago sativa. Antonie Van Leeuwenhoek 73, 3–8.
Crossref | GoogleScholarGoogle Scholar | CAS | PubMed | open url image1

Paffetti D, Scotti C, Gnocchi S, Fancelli S, Bazzicalupo M (1996) Genetic diversity of an Italian Rhizobium meliloti population from Medicago sativa varieties. Applied and Environmental Microbiology 62, 2279–2285.
CAS | PubMed |
open url image1

Palmer KM, Young JPW (2000) Higher diversity of Rhizobium leguminosarum Biovar viciae populations in arable soils than in grass soils. Applied and Environmental Microbiology 66, 2445–2450.
Crossref | GoogleScholarGoogle Scholar | CAS | PubMed | open url image1

Provorov NA, Simarov BV (1990) Genetic variation in alfalfa, sweet clover and fenugreek for the activity of symbiosis with Rhizobium meliloti. Plant Breeding 105(4), 300–310.
Crossref | GoogleScholarGoogle Scholar | open url image1

Roumiantseva ML, Andronov EE, Sharypova LA, Dammann-Kalinowski T, Keller M, Young JPW, Simarov BV (2002) Diversity of Sinorhizobium meliloti from the central Asian alfalfa gene center. Applied and Environmental Microbiology 68, 4694–4697.
Crossref | GoogleScholarGoogle Scholar | CAS | PubMed | open url image1

Seklani H, Hassen H (1990) Contribution à l’étude des espèces spontanées du genre Medicago en Tunisie. Annales de l’INRAT 63, 3–15. open url image1

Selge A , Higuchi S (2002) ‘Increasing the white clover symbiotic N2 fixation activity. Lowland and Grasslands of Europe: Utilization and Development.’ (FAO: Rome)

van Berkum P, Elia P, Eardly BD (2006) Multilocus sequence typing as an approach for population analysis of Medicago-nodulating rhizobia. Journal of Bacteriology 188, 5570–5577.
Crossref | GoogleScholarGoogle Scholar | CAS | PubMed | open url image1

Velázquez E, Mateos PF, Velasco N, Santos F, Burgos PA, Villadas P, Toro N, Martínez-Molina E (1999) Symbiotic characteristics and selection of autochthonous strains of Sinorhizobium meliloti populations in different soils. Soil Biology & Biochemistry 47, 495–502. open url image1

Vincent JM (1970) ‘A manual for practical study of root-nodule bacteria.’ IBP handbook 15. (Blackwell Scientific Publications: Oxford, UK)

Weisburg WG, Barns SM, Pelletier DA, Lane DJ (1991) 16S ribosomal DNA amplification for phylogenetic study. Journal of Bacteriology 173, 697–703.
CAS | PubMed |
open url image1

Zribi K, Mhamdi R, Huguet T, Aouani ME (2004) Distribution and genetic diversity of Rhizobium nodulating natural populations of Medicago truncatula in Tunisian soils. Soil Biology & Biochemistry 36, 903–908.
Crossref | GoogleScholarGoogle Scholar | CAS | open url image1

Zribi K, Mhamdi R, Huguet T, Aouani ME (2005) Diversity of Sinorhizobium meliloti and S. medicae nodulating Medicago truncatula according to host and soil origins. World Journal of Microbiology & Biotechnology 21, 1009–1015.
Crossref | GoogleScholarGoogle Scholar | CAS | open url image1