Abstract
Red clover (Trifolium pratense L.) is used in the improvement of grasslands in Uruguay and has been inoculated with commercial strain U204 of Rhizobium leguminosarum bv trifolii since 1970s. Native-naturalized rhizobia strains present in soil are the basis for selecting and developing new inoculants. With this aim, we evaluated the diversity of red clover rhizobia in Uruguayan red clover pastures both historically inoculated with U204 and non-inoculated ones. Thirty-eight different enterobacterial repetitive intergenic consensus (ERIC) PCR genomic fingerprints were identified, albeit surprisingly only one of 80 isolates showed an ERIC profile similar to U204. Under controlled conditions, red clover plants inoculated with one of the native isolates, strain 317, produced more biomass than those inoculated with the commercial U204. ERIC-PCR was also used to show that strain 317 competed for nodulation better than U204 in a field with previous history of inoculation. Moreover, both U204 and 317 were tagged with a gusA reporter gene and their competitiveness for nodulation assessed in various soil types. Again, strain 317 appeared more competitive than U204, particularly in soils with previous history of inoculation. Our results reinforce the long-known idea of assessing the actual needs of inoculation of legumes in different soils and suggest that the indigenous isolate 317 is an effective and competitive strain that can be used for development of a new red clover inoculant.
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References
Agius F, Sanguinetti C, Monza J (1997) Strain-specific fingerprints of Rhizobium loti generated by PCR with arbitrary and repetitive sequences. FEMS Microbiol Ecol 24:87–92
Anyango B, Wilson K, Giller K (1998) Competition in Kenyan soils between Rhizobium leguminosarum bv. phaseoli Kim5 and R. tropici strain CIAT899 using the gusA marker gene. Plant Soil 204:69–78
Batista L, Tomasco I, Lorite MJ, Sanjuán J, Monza J (2013) Diversity and phylogeny of rhizobial strains isolated from Lotus uliginosus grown in Uruguayan soils. Appl Soil Ecol 66:19–28
Beringer JE (1974) Factor de transferencia en Rhizobium leguminosarum. Gen Microbiol 84:188–198
Date RA (2000) Inoculated legumes in cropping systems of the tropics. Field Crop Res 65:123–136
de Bruijn F (1992) Use of Repetitive (repetitive extragenic palindromic and enterobacterial repetitive intergeneric consensus) sequences and the polymerase chain reaction to fingerprint the genomes of Rhizobium meliloti isolates and other soil bacteria. Appl Environ Microbiol 58:2180–2187
Dowling DN, Broughton WJ (1986) Competition for nodulation of legumes. Annu Rev Microbiol 40:131–157
Estrella MJ, Muñoz S, Soto MJ, Ruiz O, Sanjuán J (2009) Genetic diversity and host range of rhizobia nodulating Lotus tenuis in typical soils of the Salado River Basin (Argentina). Appl Environ Microbiol 75:1088–1098
Fisher RA, Yates F (1963) In: Fisher RA, Yates F (eds) tables for biological, agricultural and medical research, 6th edn. Oliver and Boyd, London
Handberg K, Stougaard J (1992) Lotus japonicus, an autogamous, diploid legume species for classical and molecular genetics. Plant J 2:487–496
Herrera-Cervera JA, Caballero-Mellado J, Laguerre G, Tichy HV, Requena N, Amarger N, Martínez-Romero E, Olivares J, Sanjuán J (1999) At least five rhizobial species nodulate Phaseolus vulgaris in a Spanish soil. FEMS Microbiol Ecol 30:87–97
Howieson JG, Yates RJ, O’Hara GW, Ryder M, Real D (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 45:199–207
Hungria M, Nogueira MA, Silva Araujo R (2013) Co-inoculation of soybeans and common beans with rhizobia and azospirilla: strategies to improve sustainability. Biol Fertil Soils 49:791–801
Laguerre G, Allard M, Revoy F, Amarger N (1994) Rapid identification of rhizobia by restriction fragment length polymorphism analysis of PCR-amplified 16S rRNA genes. Appl Environ Microbiol 60:56–63
Lindström K, Murwira M, Willems A, Altier N (2010) The biodiversity of beneficial microbe-host mutualism: the case of rhizobia. Res Microbiol 161:453–463
Lorite M, Videira e Castro I, Muñoz S, Sanjuán J (2012) Phylogenetic relationship of Lotus uliginosus symbionts with bradyrhizobia nodulating genistoid legumes. FEMS Microbiol Ecol 79:454–474
Loureiro MF, Kaschuk G, Alberton O, Hungría M (2007) Soybean [Glycine max (L.) Merrill] rhizobia diversity in Brazilian oxisols under various soil, cropping, and inoculation management. Biol Fertil Soils 43:665–674
MGAP (Ministerio de Ganadería Agricultura y Pesca). (2008) Departamento de Microbiología de Suelos. Colección Nacional de Cepas de Rhizobium spp.
Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory. 466 p
Mrabet M, Mhamdi R, Tajini F, Tiwari R, Trabelsi M, Aouani ME (2005) Competitiveness and symbiotic effectiveness of a R. gallicum strain isolated from root nodules of Phaseolus vulgaris. Eur J Agron 22:209–216
Olivares J, Bedmar EJ, Sanjuan J (2013) Biological nitrogen fixation in the context of global change. Mol Plant Microbe Interact 26:486–494
Qiu M, Li S, Zhou X, Cui X, Vivanco JM, Zhang N, Shen Q, Zhang R (2014) De-coupling of root–microbiome associations followed by antagonist inoculation improves rhizosphere soil suppressiveness. Biol Fertil Soils 50:217–224
Rebuffo M, Bemhaja M, Risso D (2006) Utilization of forage legumes in pastoral systems: state of art in Uruguay. Lotus Newsletter 36: 22–33. http://www.inia.org.uy/sitios/lnl/indexvol361.html (Accessed June 17th, 2014)
Rivas R, Velázquez E, Valverde A, Mateos PF, Martínez-Molina E (2001) A two primers random amplified polymorphic DNA procedure to obtain polymerase chain reaction fingerprints of bacterial species. Electrophoresis 22:1086–1089
Rodríguez-Blanco A, Sicardi M, Frioni L (2010) Competition for nodule occupancy between introduced and native strains of Rhizobium leguminosarum biovar trifolii. Biol Fertil Soils 46:419–425
Rufini M, Pereira da Silva MA, Avelar Ferreira PA, de Souza CA, Lima Soares B, de Andrade MJB, de Souza Moreira FM (2014) Symbiotic efficiency and identification of rhizobia that nodulate cowpea in a Rhodic Eutrudox. Biol Fertil Soils 50:115–132
Sessitsch A, Hardarson G, de Vos WM, Wilson KJ (1998) Use of marker genes in competition studies of Rhizobium. Plant Soil 204:35–45
Simon T, Salava J (2006) New Rhizobium leguminosarum bv. trifolii isolates: evaluation of competitiveness for clover nodule occupancy. Plant Soil Environ 52:441–448
Streit W, Botero L, Werner D, Beck D (1995) Competition for nodule occupancy on Phaseolus vulgaris by Rhizobium etli and Rhizobium tropici strains can be efficiently monitored in an ultisol during the early stages of growth using a constitutive GUS gene fusion. Soil Biol Biochem 27:1075–1081
Thies JE, Bohlool BB, Singleton PW (1992) Environmental effects on nodulation for nodule occupancy between introduced and indigenous rhizobia and among introduced strains. Can J Microbiol 38:493–500
Toro N (1996) Nodulation competitiveness in the Rhizobium-legume symbiosis. World J Microbiol Biotechnol 12:57–162
Triplett E, Sadowsky M (1992) Genetic of competition for nodulation of legumes. Annu Rev Microbiol 46:399–428
Vincent J (1970) A manual for the practical study of root-nodule bacteria. IBP Handbook 15. Blackwell Scientific Publications, Oxford, p 6
Wilson KJ, Giller KE, Jefferson RA (1991) P-glucuronidase (GUS) operon fusions as a tool for studying plant-microbe interactions. In: Hennecke H, Verma DPS, Hennecke H, Verma DPS (eds) Advances in molecular genetics of plant-microbe interactions, vol. 1. Kluwer Academic Publishers, Dordrecht, pp 226–229
Wilson K, Sessitsch A, Corbo J, Giller K, Akkermans A, Jefferson R (1995) β-Glucuronidase (GUS) transposons for ecological and genetic studies of rhizobia and other Gram-negative bacteria. Microbiology 141:1691–1705
Yates RJ, Howieson JG, Real D, Reeve WG, Vivas-Marfisi A, O’Hara GW (2005) Evidence of selection for effective nodulation in the Trifolium spp. symbiosis with Rhizobium leguminosarum biovar trifolii. Aust J Exp 45:189–198
Acknowledgments
Authors are grateful to the funding of grants from FONTAGRO FTG-787/2005 (LESIS), the European LOTASSA project (FP6-2005-INCO-DEV2-517617), and ANII (Magister grant for L.B.). We also thank M. Sánchez for helping with laboratory assays and R. Zarza for soil sampling.
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Batista, L., Irisarri, P., Rebuffo, M. et al. Nodulation competitiveness as a requisite for improved rhizobial inoculants of Trifolium pratense . Biol Fertil Soils 51, 11–20 (2015). https://doi.org/10.1007/s00374-014-0946-3
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DOI: https://doi.org/10.1007/s00374-014-0946-3