Skip to main content

Microbial Inoculants with Autochthonous Bacteria for Biodiverse Legume Pastures in Portuguese Agro-Forestry Ecosystems

  • Conference paper
  • First Online:
Biological Nitrogen Fixation and Beneficial Plant-Microbe Interaction

Abstract

One key point to achieve the success of microorganisms like rhizobia as biofertilizers (inoculants) is the characterization and selection of autochthonous strains. The aim of this work was to study the nitrogen-fixing efficiency of autochthonous Rhizobium strains, using as hosts several agronomic important annual Trifolium species (Trifolium subterraneum, Trifolium incarnatum, Trifolium suaveolens, and Trifolium vesiculosum), and compare them with a peat commercial inoculant . For this purpose, three preselected strains (89Ts2a, 123Ts2a, and 149Ts2) isolated from several Trifolium spp. grown in the south of Portugal were characterized beyond their symbiotic performance, also for their “in vitro” PPB activities (solubilization of mineral phosphate and production of siderophores). In addition, molecular identification was performed using the sequence of 16S rRNA and recA genes. Results of shoot dry weight indicated that autochthonous Rhizobium strains were highly efficient in nitrogen fixation. For T. subterraneum and T. incarnatum, strain 123TS2a had the best performance, and for T. suaveolens and T. vesiculosum, the best performance was accomplished by strain 89TS2a. These two strains were combined in a mixed inoculum that was shown to promote higher yields than the commercial peat inoculant in the majority of Trifolium species tested. Results of “in vitro” activities indicated that two strains (89Ts2 and 123Ts2a) could solubilize phosphate, and the three strains could produce siderophores. All these strains had strong phylogenetic relationship with Rhizobium leguminosarum and are closely related to biovar trifolii and viciae.

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

Access this chapter

Chapter
USD 29.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 169.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 219.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  • Aguilar OM, López MV, Riccillo PM (2001) The diversity of rhizobia nodulating beans in Northwest Argentina as a source of more efficient inoculant strains. J Biotechnol 91(2–3):181–188

    Article  CAS  PubMed  Google Scholar 

  • Bergersen FJ, Brockwell J, Gibson AH et al (1971) Studies of natural populations and mutants of Rhizobium in the improvement of legume inoculants. Plant Soil 35(1):3–16

    Article  Google Scholar 

  • Beringer JE (1974) R factor transfer in Rhizobium leguminosarum. J Gen Microbiol 84(1):188–198

    CAS  PubMed  Google Scholar 

  • Brasier C (1996) Phytophthora cinnamomi and oak decline in southern Europe. Environmental constraints including climate change. Ann For Sci 53(2–3):347–358

    Google Scholar 

  • Brockwell J, Gault R, Zorin M et al (1982) Effects of environmental variables on the competition between inoculum strains and naturalized populations of Rhizobium trifolii for nodulation of Trifolium subterraneum L. and on rhizobia persistence in the soil. Aust J Agric Res 33(5):803

    Article  Google Scholar 

  • Chatel DL, Shipton WA, Parker CA (1973) Establishment and persistence of Rhizobium trifolii in western Australian soils. Soil Biol Biochem 5(6):815–824

    Article  Google Scholar 

  • Collins MT, Thies JE, Abbott LK (2002) Diversity and symbiotic effectiveness of Rhizobium leguminosarum bv. trifolii isolates from pasture soils in south-western Australia. Aust J Soil Res 40(8):1319

    Google Scholar 

  • Crespo DG (2006) The role of pasture improvement on the rehabilitation of the montado/dehesa system and in developing its traditional products. In: Ramalho Ribeiro JMC, Horta AEM, Mosconi C et al (eds) Animal products from the Mediterranean area. The Netherlands Academic Publishers, Wageningen, pp 185–197

    Google Scholar 

  • Drew EA, Charman N, Dingemanse R et al (2011) Symbiotic performance of Mediterranean Trifolium spp. with naturalised soil rhizobia. Crop Pasture Sci 62(10):903

    Google Scholar 

  • Ezawa T, Cavagnaro TR, Smith SE et al (2004) Rapid accumulation of polyphosphate in extraradical hyphae of an arbuscular mycorrhizal fungus as revealed by histochemistry and a polyphosphate kinase/luciferase system. New Phytol 161(2):387–392

    Article  CAS  Google Scholar 

  • Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39(4):783–791

    Article  Google Scholar 

  • Ferguson BJ, Indrasumunar A, Hayashi S et al (2010) Molecular analysis of legume nodule development and autoregulation. J Integr Plant Biol 52(1):61–76

    Article  CAS  PubMed  Google Scholar 

  • Ferreira EM, Castro IV (2011) Fijación biológica de nitrógeno y productividade de pastos naturales en sistemas agrícolas del mediterráneo. In: Megías M, Rivilla R, Soto MJ et al (eds) Fundamentos y Aplicaciones agroambientales de las interacciones beneficiosas plantas-microorganismos. Sociedad Española de Fijación de Nitrógeno (SEFIN), Spain, pp 403–416

    Google Scholar 

  • Ferreira EM, Marques JF (1992) Selection of Portuguese Rhizobium leguminosarum bv. trifolii strains for production of legume inoculants. Plant Soil 147(1):151–158

    Article  Google Scholar 

  • Ferreira EM, Simões N, Castro IV et al (2010) Relationships of selected soil parameters and natural pastures yield in the Montado ecosystem of the Mediterranean area using multivariate analysis. Silva Lus 18(2):151–166

    Google Scholar 

  • Gaunt MW, Turner SL, Rigottier-Gois L et al (2001) Phylogenies of atpD and recA support the small subunit rRNA-based classification of rhizobia. Int J Syst Evol Microbiol 51(Pt 6):2037–2048

    Article  CAS  PubMed  Google Scholar 

  • Gibson AH, Curnow BC, Bergersen FJ et al (1975) Studies of field populations of Rhizobium: effectiveness of strains of Rhizobium trifolii associated with Trifolium subterraneum L. pastures in south-eastern Australia. Soil Biol Biochem 7(2):95–102

    Article  Google Scholar 

  • Glick BR (1995) The enhancement of plant growth by free-living bacteria. Can J Microbiol 41(2):109–117

    Article  CAS  Google Scholar 

  • He ZL, Bian W, Zhu J (2007) Screening and identification of microorganisms capable of utilizing phosphate adsorbed by goethite. Commun Soil Sci Plant Anal 33(5–6):647–663

    Google Scholar 

  • Howieson JG, Yates RJ, O’Hara GW et al (2005) The interactions of Rhizobium leguminosarum biovar trifolii in nodulation of annual and perennial Trifolium spp. from diverse centres of origin. Aust J Exp Agric 45(3):199

    Article  Google Scholar 

  • Huo YX, Wernick DG, Liao JC (2012) Toward nitrogen neutral biofuel production. Curr Opin Plant Biol 23(3):406–413

    CAS  Google Scholar 

  • Jensen HL (1941) Nitrogen fixation in leguminous plants. I. General characters of root-nodule bacteria isolated from species of Medicago and Trifolium in Australia. Proc Linn Soc NSW 67:98–108

    Google Scholar 

  • Kamboj D, Kumar R, Kumari A et al (2008) Rhizobia, nod factors and nodulation—a review. Agric Rev 29(3):200–206

    Google Scholar 

  • Kloepper JW, Lifshitz R, Zablotowicz RM (1989) Free-living bacterial inocula for enhancing crop productivity. Trends Biotechnol 7(2):39–44

    Article  Google Scholar 

  • Lemanceau P, Alabouvette C (1993) Suppression of fusarium wilts by fluorescent pseudomonads: mechanisms and applications. Biocontrol Sci Tech 3(3):219–234

    Article  Google Scholar 

  • Okon Y, Labandera-Gonzalez CA (1994) Agronomic applications of Azospirillum: an evaluation of 20 years worldwide field inoculation. Soil Biol Biochem 26(12):1591–1601

    Article  CAS  Google Scholar 

  • Peix A, Rivas-Boyero AA, Mateos PF et al (2001) Growth promotion of chickpea and barley by a phosphate solubilizing strain of Mesorhizobium mediterraneum under growth chamber conditions. Soil Biol Biochem 33(1):103–110

    Article  CAS  Google Scholar 

  • Pérez-Miranda S, Cabirol N, George-Téllez R et al (2007) O-CAS, a fast and universal method for siderophore detection. J Microbiol Methods 70(1):127–131

    Article  PubMed  Google Scholar 

  • Rogel MA, Ormeño-Orrillo E, Martinez Romero E (2011) Symbiovars in rhizobia reflect bacterial adaptation to legumes. Syst Appl Microbiol 34(2):96–104

    Article  PubMed  Google Scholar 

  • Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4(4):406–425

    CAS  PubMed  Google Scholar 

  • Santi C, Bogusz D, Franche C (2013) Biological nitrogen fixation in non-legume plants. Ann Bot 111(5):743–767

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Slattery JF, Coventry DR (1995) Acid-tolerance and symbiotic effectiveness of Rhizobium leguminosarum bv. trifolii isolated from subterranean clover growing in permanent pastures. Soil Biol Biochem 27(1):111–115

    Article  CAS  Google Scholar 

  • Tamura K, Peterson D, Peterson N et al (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28(10):2731–2739

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tóth G, Guicharnauda R-A, Tóth B et al (2014) Phosphorus levels in croplands of the European Union with implications for P fertilizer use. Eur J Agron 55:42–52

    Article  Google Scholar 

  • van Rhijn P, Vanderleyden J (1995) The Rhizobium-plant symbiosis. Microbiol Rev 59(1):124–142

    PubMed  PubMed Central  Google Scholar 

  • Vincent JM (1954) The root-nodule bacteria as factors in clover establishment in the red basaltic soils of the Lismore district, NSW. III. Field inoculation trials. Aust J Agric Res 5:61

    Article  CAS  Google Scholar 

  • Vincent JM (1970) A manual for the practical study of root nodule bacteria. IBP Handbook No. 15. Blackwell Scientific Publications, Oxford

    Google Scholar 

  • Weisburg WG, Barns SM, Pelletier DA et al (1991) 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 173(2):697–703

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

The financial support was provided by the project PRODER 54971 (Portugal). Ricardo Soares and Eva Arcos were granted, respectively, by PRODER 54971 (Portugal) and by FUNDECYT (Spain).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Isabel Videira e Castro .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2016 Springer International Publishing Switzerland

About this paper

Cite this paper

Soares, R., Arcos, E., Ferreira, E., Videira e Castro, I. (2016). Microbial Inoculants with Autochthonous Bacteria for Biodiverse Legume Pastures in Portuguese Agro-Forestry Ecosystems. In: González-Andrés, F., James, E. (eds) Biological Nitrogen Fixation and Beneficial Plant-Microbe Interaction. Springer, Cham. https://doi.org/10.1007/978-3-319-32528-6_15

Download citation

Publish with us

Policies and ethics