Skip to main content
SpringerLink
Log in

Alfalfa microsymbionts from different ITS and nodC lineages of Ensifer meliloti and Ensifer medicae symbiovar meliloti establish efficient symbiosis with alfalfa in Spanish acid soils

  • Environmental biotechnology
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

Alfalfa (Medicago sativa L.) is an important crop worldwide whose cropping in acid soils is hampered by the poor nodulation and yield commonly attributed to the sensitivity of its endosymbionts to acid pH. In this work, we isolated several acid-tolerant strains from alfalfa nodules in three acid soils in northwestern Spain. After grouping by RAPD fingerprinting, most strains were identified as Ensifer meliloti and only two strains as Ensifer medicae according to their 16S–23S intergenic spacer (ITS) sequences that allowed the differentiation of two groups within each one of these species. The two ITS groups of E. meliloti and the ITS group I of E. medicae have been previously found in Medicago nodules; however, the group II of E. medicae has been only found to date in Prosopis alba nodules. The analysis of the nodC gene showed that all strains isolated in this study belong to the symbiovar meliloti, grouping with the type strains of E. meliloti or E. medicae, but some harboured nodC gene alleles different from those found to date in alfalfa nodules. The strains of E. medicae belong to the symbiovar meliloti which should be also recognised in this species, although they harboured a nodC allele phylogenetically divergent to those from E. meliloti strains. Microcosm experiments showed that inoculation of alfalfa with selected acid-tolerant strains significantly increased yields in acid soils representing a suitable agricultural practice for alfalfa cropping in these soils.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  • Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410

    Article  CAS  PubMed  Google Scholar 

  • Brockwell J, Pilka A, Holliday RA (1991) Soil-pH is a major determinant of the numbers of naturally-occurring Rhizobium meliloti in noncultivated soils in Central New-South-Wales. Aust J Exp Agr 31:211–219

    Article  Google Scholar 

  • Capela D, Barloy-Hubler F, Gouzy J, Bothe G, Ampe F, Batut J, Boistard P, Becker A, Boutry M, Cadieu E, Dréano S, Gloux S, Godrie T, Goffeau A, Kahn D, Kiss E, Lelaure V, Masuy D, Pohl T, Portetelle D, Pühler A, Purnelle B, Ramsperger U, Renard C, Thébault P, Vandenbol M, Weidner S, Galibert F (2001) Analysis of the chromosome sequence of the legume symbiont Sinorhizobium meliloti strain 1021. Proc Natl Acad Sci U S A 98:9877–9882

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Charman N, Ballard RA, Humphries AW, Auricht GC (2008) Improving lucerne nodulation at low pH: contribution of rhizobial and plant genotype to the nodulation of lucerne seedlings growing in solution culture at pH 5. Aust J Exp Agr 48:512–517

    Article  CAS  Google Scholar 

  • Cobo-Díaz JF, Martínez-Hidalgo P, Fernández-González AJ, Martínez-Molina E, Toro N, Velázquez E, Fernández-López M (2014) The endemic Genista versicolor from sierra Nevada national park in Spain is nodulated by putative new Bradyrhizobium species and a novel symbiovar (sierranevadense). Syst Appl Microbiol 37:177–185

    Article  PubMed  Google Scholar 

  • Del Papa MF, Pistorio M, Balague LJ, Draghi WO, Wegener C, Perticari A, Niehaus K, Lagares A (2003) A microcosm study on the influence of pH and the host-plant on the soil persistence of two alfalfa-nodulating rhizobia with different saprophytic and symbiotic characteristics. Biol Fertil Soils 39:112–116

    Article  Google Scholar 

  • del Villar M, Rivas R, Peix A, Mateos PF, Martínez-Molina E, van Berkum P, Willems A, Velázquez E (2008) Stable low molecular weight RNA profiling showed variations within Sinorhizobium meliloti and Sinorhizobium medicae nodulating different legumes from the alfalfa cross-inoculation group. FEMS Microbiol Lett 282:273–281

    Article  PubMed  Google Scholar 

  • Djedidi S, Yokoyama T, Tomooka N, Ohkama-Ohtsu N, Risal CP, Abdellye C, Sekimoto H (2011) Phenotypic and genetic characterization of rhizobia associated with alfalfa in the Hokkaido and Ishigaki regions of Japan. Syst Appl Microbiol 34:453–461

    Article  PubMed  Google Scholar 

  • Elboutahiri N, Thami-Alami I, Udupa SM (2010) Phenotypic and genetic diversity in Sinorhizobium meliloti and S. medicae from drought and salt affected regions of Morocco. BMC Microbiol 10:15

    Article  PubMed Central  PubMed  Google Scholar 

  • Faghire M, Mandri B, Oufdou K, Bargaz A, Ghoulam C, Ramírez-Bahena MH, Velázquez E, Peix A (2012) Identification at the species and symbiovar levels of strains nodulating Phaseolus vulgaris in saline soils of the Marrakech region (Morocco) and analysis of the otsA gene putatively involved in osmotolerance. Syst Appl Microbiol 35:156–164

    Article  CAS  PubMed  Google Scholar 

  • Ferreira L, Sánchez-Juanes F, García-Fraile P, Rivas R, Mateos PF, Martínez-Molina E, González-Buitrago JM, Velázquez E (2011) MALDI-TOF mass spectrometry is a fast and reliable platform for identification and ecological studies of species from family Rhizobiaceae. PLoS ONE 6(5):15

    Google Scholar 

  • Galardini M, Pini F, Bazzicalupo M, Biondi EG, Mengoni A (2013) Replicon-dependent bacterial genome evolution: the case of Sinorhizobium meliloti. Genome Biol Evol 5:542–558

    Article  PubMed Central  PubMed  Google Scholar 

  • Glenn AR, Dilworth MJ (1994) The life of root-nodule bacteria in the acidic underground. FEMS Microbiol Lett 123:1–9

    Article  CAS  Google Scholar 

  • Gubry-Rangin C, Béna G, Cleyet-Marel JC, Brunel B (2013) Definition and evolution of a new symbiovar, sv. rigiduloides, among Ensifer meliloti efficiently nodulating Medicago species. Syst Appl Microbiol 36:490–496

    Article  PubMed  Google Scholar 

  • Guo JH, Liu XJ, Zhang Y, Shen JL, Han WX, Zhang WF, Christie P, Goulding KWT, Vitousek PM, Zhang FS (2010) Significant acidification in major Chinese croplands. Science 327:1008–1010

    Article  CAS  PubMed  Google Scholar 

  • Helrich K (1990). Fertilizers. In: Helrich K (ed) Methods of analysis of the Association of Official Analytical Chemists. Fifteenth edition, vol 1. AOAC, Arlington, VA, pp. 9–39

  • Howieson JG, Ewing MA, Dantuono MF (1988) Selection for acid tolerance in Rhizobium meliloti. Plant Soil 105:179–188

    Article  CAS  Google Scholar 

  • Iglesias O, Rivas R, García-Fraile P, Abril A, Mateos PF, Martínez-Molina E, Velázquez E (2007) Genetic characterization of fast-growing rhizobia able to nodulate Prosopis alba in North Spain. FEMS Microbiol Lett 277:210–216

    Article  CAS  PubMed  Google Scholar 

  • Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16:111–120

    Article  CAS  PubMed  Google Scholar 

  • Laguerre G, Nour SM, Macheret V, Sanjuán 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-UK 147:981–993

    CAS  Google Scholar 

  • Langer H, Nandasena KG, Howieson JG, Jorquera M, Borie F (2008) Genetic diversity of Sinorhizobium meliloti associated with alfalfa in Chilean volcanic soils and their symbiotic effectiveness under acidic conditions. World J Microbiol Biotechnol 24:301–308

    Article  Google Scholar 

  • Martínez-Abarca F, Martínez-Rodríguez L, López-Contreras JA, Jiménez-Zurdo JI, Toro N (2013) Complete genome sequence of the alfalfa symbiont Sinorhizobium/Ensifer meliloti strain GR4. Genome Announc 1(1):e00174–12

    Article  PubMed Central  PubMed  Google Scholar 

  • Mnasri B, Mrabet M, Laguerre G, Aouani ME, Mhamdi R (2007) 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. Arch Microbiol 187:79–85

    Article  CAS  PubMed  Google Scholar 

  • Mnasri B, Badri Y, Saidi S, de Lajudie P, Mhamdi R (2009) Symbiotic diversity of Ensifer meliloti strains recovered from various legume species in Tunisia. Syst Appl Microbiol 32:583–592

    Article  CAS  PubMed  Google Scholar 

  • Moschetti G, Peluso A, Protopapa A, Anastasio M, Pepe O, Defez R (2005) Use of nodulation pattern, stress tolerance, nodC gene amplification, RAPD-PCR and RFLP-16S rDNA analysis to discriminate genotypes of Rhizobium leguminosarum biovar viciae. Syst Appl Microbiol 28:619–631

    Article  CAS  PubMed  Google Scholar 

  • Mulas D, García-Fraile P, Carro L, Ramírez-Bahena MH, Casquero P, Velázquez E, González-Andrés F (2011) Distribution and efficiency of Rhizobium leguminosarum strains nodulating Phaseolus vulgaris in Northern Spanish soils: selection of native strains that replace conventional N fertilization. Soil Biol Biochem 43:2283–2293

    Article  CAS  Google Scholar 

  • Munns DN (1968) Nodulation of Medicago sativa in solution culture. I. Acid-sensitive steps. Plant Soil 28:129–146

    Article  Google Scholar 

  • Paffetti D, Scotti C, Gnocchi S, Fancelli S, Bazzicalupo M (1996) Genetic diversity of an Italian Rhizobium meliloti population from different Medicago sativa varieties. Appl Environ Microbiol 62:2279–2285

    PubMed Central  CAS  PubMed  Google Scholar 

  • Peix A, Ramírez-Bahena MH, Velázquez E, Bedmar EJ (2015) Bacterial associations with legumes. Crit Rev Plant Sci 34:17–42

    Article  Google Scholar 

  • Perret X, Staehelin C, Broughton WJ (2000) Molecular basis of symbiotic promiscuity. Microbiol Mol Biol Rev 64:180–201

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Pinkerton A, Simpson J (1986) Responses of some crop plants to correction of subsoil acidity. Aust J Exp Agr 26:107–113

    Article  Google Scholar 

  • Ramírez-Bahena MH, Chahboune R, Velázquez E, Gomez-Moriano A, Mora E, Peix A, Toro M (2013) Centrosema is a promiscuous legume nodulated by several new putative species and symbiovars of Bradyrhizobium in various American countries. Syst Appl Microbiol 36(6):392–400

    Article  PubMed  Google Scholar 

  • Reeve W, Chain P, O’Hara G, Ardley J, Nandesena K, Breu L, Tiwari R, Malfatti S, Kiss H, Lapidus A, Copeland A, Nolan M, Land M, Hauser L, Chang YJ, Ivanova N, Mavromatis K, Markowitz V, Kyrpides N, Gollagher M, Yates R, Dilworth M, Howieson J (2010) Complete genome sequence of the Medicago microsymbiont Ensifer (Sinorhizobium) medicae strain WSM419. Stand Genomic Sci 2:77–86

    Article  PubMed Central  PubMed  Google Scholar 

  • Rice WA, Penney DC, Nyborg M (1977) Effects of soil acidity on rhizobia numbers, nodulation and nitrogen-fixation by alfalfa and red-clover. Can J Soil Sci 57:197–203

    Article  CAS  Google Scholar 

  • Rigaud J, Puppo A (1975) Indole-3-acetic-acid catabolism by soybean bacteroids. J Gen Microbiol 88:223–228

    Article  Google Scholar 

  • Rivas R, García-Fraile P, Mateos PF, Martínez-Molina E, Velázquez E (2007a) Characterization of xylanolytic bacteria present in the bract phyllosphere of the date palm Phoenix dactylifera. Lett Appl Microbiol 44:181–187

    Article  CAS  PubMed  Google Scholar 

  • Rivas R, Laranjo M, Mateos PF, Oliveira S, Martínez-Molina E, Velázquez E (2007b) Strains of Mesorhizobium amorphae and Mesorhizobium tianshanense, carrying symbiotic genes of common chickpea endosymbiotic species, constitute a novel biovar (ciceri) capable of nodulating Cicer arietinum. Lett Appl Microbiol 44:412–418

    Article  CAS  PubMed  Google Scholar 

  • Rogel MA, Ormeño-Orrillo E, Martínez-Romero E (2011) Symbiovars in rhizobia reflect bacterial adaptation to legumes. Syst Appl Microbiol 34:96–104

    Article  PubMed  Google Scholar 

  • 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. Appl Environ Microbiol 68:4694–4697

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–25

    CAS  PubMed  Google Scholar 

  • Sallet E, Roux B, Sauviac L, Jardinaud MF, Carrère S, Faraut T, de Carvalho-Niebel F, Gouzy J, Gamas P, Capela D, Bruand C, Schiex T (2013) Next-generation annotation of prokaryotic genomes with EuGene-P: application to Sinorhizobium meliloti 2011. DNA Res 20:339–353

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Segundo E, Martínez-Abarca F, van Dillewijn P, Fernández-López M, Lagares A, Martínez-Drets G, Niehaus K, Pühler A, Toro N (1999) Characterisation of symbiotically efficient alfalfa-nodulating rhizobia isolated from acid soils of Argentina and Uruguay. FEMS Microbiol Ecol 28:169–176

    Article  CAS  Google Scholar 

  • Silva C, Kan FL, Martínez-Romero E (2007) Population genetic structure of Sinorhizobium meliloti and S. medicae isolated from nodules of Medicago spp. in Mexico. FEMS Microbiol Ecol 60:477–489

    Article  CAS  PubMed  Google Scholar 

  • Stanley M, Britton RS, Christinat RA (2002) Success with lucerne. PIRSA Publishing Services, Topcrop

    Google Scholar 

  • Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • van Berkum P, Elia P, Eardly BD (2010) application of multilocus sequence typing to study the genetic structure of megaplasmids in Medicago-nodulating rhizobia. Appl Environ Microbiol 76:3967–3977

    Article  PubMed Central  PubMed  Google Scholar 

  • Villegas MD, Rome S, Maure L, Domergue O, Gardan L, Bailly X, Cleyet-Marel JC, Brunel B (2006) Nitrogen-fixing sinorhizobia with Medicago laciniata constitute a novel biovar (bv. medicaginis) of S. meliloti. Syst Appl Microbiol 29:526–538

    Article  Google Scholar 

  • Vincent JM (1970) The cultivation, isolation and maintenance of rhizobia. In: Vincent JM (ed) A manual for the practical study of root-nodule bacteria. Blackwell Scientific Publications, Oxford, pp 1–13

    Google Scholar 

  • Weidner S, Baumgarth B, Göttfert M, Jaenicke S, Pühler A, Schneiker-Bekel S, Serrania J, Szczepanowski R, Becker A (2013) Genome sequence of Sinorhizobium meliloti Rm41. Genome Announc 1(1):e00013–12

    Article  PubMed Central  PubMed  Google Scholar 

  • Zurdo-Piñeiro JL, García-Fraile P, Rivas R, Peix A, León-Barrios M, Willems A, Mateos PF, Martínez-Molina E, Velázquez E, van Berkum P (2009) Rhizobia from Lanzarote, the Canary Islands, that nodulate Phaseolus vulgaris have characteristics in common with Sinorhizobium meliloti isolates from mainland Spain. Appl Environ Microbiol 75:2354–2359

    Article  PubMed Central  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by Junta de Castilla y León (JCyL, Spanish Regional Government) grant CSI02A09. MHRB is recipient of a JAE-Doc research contract of CSIC (Spain) cofinanced by ERDF. We thank M. Faghire for his valuable help in statistic analyses and A. Gómez-Moriano for technical assistance.

Author information

Authors and Affiliations

  1. Instituto de Recursos Naturales y Agrobiología (IRNASA-CSIC), Cordel de Merinas 40-52, 37008, Salamanca, Spain

    Martha-Helena Ramírez-Bahena, Margarita Vargas, María Martín & Álvaro Peix

  2. Unidad Asociada Grupo de Interacción Planta-Microorganismo, Universidad de Salamanca-IRNASA-CSIC, Salamanca, Spain

    Martha-Helena Ramírez-Bahena, Encarna Velázquez & Álvaro Peix

  3. Departamento de Microbiología y Genética, Universidad de Salamanca, Salamanca, Spain

    Carmen Tejedor & Encarna Velázquez

Authors
  1. Martha-Helena Ramírez-Bahena
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Margarita Vargas
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. María Martín
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Carmen Tejedor
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Encarna Velázquez
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Álvaro Peix
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Álvaro Peix.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ramírez-Bahena, MH., Vargas, M., Martín, M. et al. Alfalfa microsymbionts from different ITS and nodC lineages of Ensifer meliloti and Ensifer medicae symbiovar meliloti establish efficient symbiosis with alfalfa in Spanish acid soils. Appl Microbiol Biotechnol 99, 4855–4865 (2015). https://doi.org/10.1007/s00253-014-6347-6

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-014-6347-6

Keywords

Search

Navigation