Identification of soybean Bradyrhizobium strains used in commercial inoculants in Brazil by MALDI-TOF mass spectrometry

A Correction to this article was published on 14 August 2019

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Abstract

Biological nitrogen fixation (BNF) with the soybean crop probably represents the major sustainable technology worldwide, saving billions of dollars in N fertilizers and decreasing water pollution and the emission of greenhouse gases. Accordingly, the identification of strains occupying nodules under field conditions represents a critical step in studies that are aimed at guaranteeing increased BNF contribution. Current methods of identification are mostly based on serology, or on DNA profiles. However, the production of antibodies is restricted to few laboratories, and to obtain DNA profiles of hundreds of isolates is costly and time-consuming. Conversely, the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS technique might represent a golden opportunity for replacing serological and DNA-based methods. However, MALDI-TOF databases of environmental microorganisms are still limited, and, most importantly, there are concerns about the discrimination of protein profiles at the strain level. In this study, we investigated four soybean rhizobial strains carried in commercial inoculants used in over 35 million hectares in Brazil and also in other countries of South America and Africa. A supplementary MALDI-TOF database with the protein profiles of these rhizobial strains was built and allowed the identification of unique profiles statistically supported by multivariate analysis and neural networks. To test this new database, the nodule occupancy by Bradyrhizobium strains in symbiosis with soybean was characterized in a field experiment and the results were compared with serotyping of bacteria by immuno-agglutination. The results obtained by both techniques were highly correlated and confirmed the viability of using the MALDI-TOF MS technique to effectively distinguish bacteria at the strain level.

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Change history

  • 14 August 2019

    The original version of this article unfortunately contained a mistake. The presentation of Fig. 1was incorrect. The correct version is given below.

References

  1. 1.

    Andrade A, Pinto SC, Oliveira RS (2002) Animais de Laboratório: criação e experimentação. FIOCRUZ, Rio de Janeiro

  2. 2.

    Boddey LH, Hungria M (1997) Phenotypic grouping of Brazilian Bradyrhizobium strains which nodulate soybean. Biol Fertil Soils 25:407–415

    CAS  Article  Google Scholar 

  3. 3.

    Calderaro A, Arcangeletti MC, Rodighiero I, Buttrini M, Gorrini C, Motta F, Germini D, Medici MC, Chezzi C, De Conto F (2014) Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry applied to virus identification. Sci Rep 4:1–10

    Google Scholar 

  4. 4.

    Chang WS, Lee HI, Hungria M (2015) Soybean production in the Americas. In: Lugtenberg B (ed) Principles of plant-microbe interactions. Springer International Publishing, Basel, pp 393–400

    Google Scholar 

  5. 5.

    Delamuta JRR, Ribeiro RA, Ormeño-Orrilho E, Melo IS, Martínez-Romero E, Hungria M (2013) Polyphasic evidence supporting the reclassification of Bradyrhizobium japonicum group Ia strains as Bradyrhizobium diazoefficiens sp. nov. Int J Syst Evol Microbiol 63:3342–3351

    CAS  Article  Google Scholar 

  6. 6.

    Dowling DN, Broughton WJ (1986) Competition for nodulation of legumes. Annu Rev Microbiol 40:191–157

    Article  Google Scholar 

  7. 7.

    Fenselau C, Demirev PA (2001) Characterization of intact microorganisms by MALDI mass spectrometry. Mass Spectrom Rev 20:157–171

    CAS  Article  Google Scholar 

  8. 8.

    Ferreira L, Saánchez-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:e20223. https://doi.org/10.1371/journal.pone.0020223

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Ham GE, Frederick LR, Anderson IC (1971) Serogroups of Rhizobium japonicum in soybean nodules samples in Iowa. Agron J 63:69–72

    Article  Google Scholar 

  10. 10.

    Hungria M, Vargas MAT (1996) Exploring the microbial diversity and soil management practices to optimize the contribution of soil microorganisms to plant nutrition. In: Stacey G, Mullin B, Gresshoff PM (eds). Biology of plant-microbe interactions. International Society of Molecular Plant-Microbe Interactions, St. Paul, 493–496

  11. 11.

    Hungria M (1994) Coleta de nódulos e isolamento de rizóbios. In: Hungria M, Araújo RS (eds). Manual de métodos empregados em estudos de microbiologia agrícola. EMBRAPA, Brasília, 157–170

  12. 12.

    Hungria M, Campo RJ, Mendes IC (2003) Benefits of inoculation of the common bean (Phaseolus vulgaris) crop with efficient and competitive Rhizobium tropici strains. Biol Fertil Soils 39:88–93

    Article  Google Scholar 

  13. 13.

    Hungria M, Mendes IC (2015) Nitrogen fixation with soybean: the perfect symbiosis? In: De Bruijn FJ (ed) Biological nitrogen fixation vol. 2. John Wiley and Sons Inc., New Jersey, pp 1005–1019

    Google Scholar 

  14. 14.

    Joliffe IT (2002) Principal component analysis, 2nd edition. Springer-Verlag, New York

    Google Scholar 

  15. 15.

    Keller PM, Bruderer V, Müller F (2016) Restricted identification of clinical pathogens categorized as biothreats by MALDI-TOF mass spectrometry. J Clin Microbiol 54:816

    CAS  Article  Google Scholar 

  16. 16.

    Lavigne JP, Espinal P, Messad N, Pantel A, Sotto A (2013) Mass spectrometry: a revolution in clinical microbiology? Clin Chem Lab Med 51:257–270

    CAS  Article  Google Scholar 

  17. 17.

    Lima SC, Lopes ES, Lemos EGM (1998) Caracterização de rizóbios (Bradyrhizobium japonicum) e produtividade de soja. Sci Agric 55:45–69

    Google Scholar 

  18. 18.

    McLoughlin TJ, Alt SG, Gonzalez RG, Romero-Severson J (1991) Effects on soybean seed yield by members of 123 serocluster and USDA 110 in the greenhouse and in soils low in indigenous Bradyrhizobium japonicum. Can J Microbiol 37:984–988

    Article  Google Scholar 

  19. 19.

    Mendes IC, Vargas MAT, Hungria M (2004) Establishment of Bradyrhizobium japonicum and B. elkanii in a Brazilian Cerrados Oxisol. Biol Fertil Soils 40:28–35

    Article  Google Scholar 

  20. 20.

    Menna P, Barcellos FG, Hungria M (2009) Phylogeny and taxonomy of a diverse collection of Bradyrhizobium strains based on multilocus sequence analysis of 16S rRNA, ITS, glnII, recA, atpD and dnaK genes. Int J Syst Evol Microbiol 59:2934–2950

    CAS  Article  Google Scholar 

  21. 21.

    Mpepereki S, Wollum AG (1991) Diversity of indigenous Bradyrhizobium japonicum in North Carolina soils. Biol Fertil Soils 11:121–127

    Article  Google Scholar 

  22. 22.

    Ndungu SM, Messmer MM, Ziegler D, Thuita M, Vanlauwe B, Frossard E, Thonar C (2018) Evaluation of MALDI-TOF mass spectrometry for the competitiveness analysis of selected indigenous cowpea (Vigna unguiculata L. Walp.) Bradyrhizobium strains from Kenya. Appl Microbiol Biotechnol 102:5265–5278

    CAS  Article  Google Scholar 

  23. 23.

    Peres JRR, Mendes IC, Suhet AR, Vargas MAT (1993) Eficiência e competitividade de estirpes de rizóbio para a soja em solos de Cerrados. Rev Bras Cienc Solo 17:357–363

  24. 24.

    Peres JRR, Vidor C (1980) Seleção de estirpes de Rhizobium japonicum e competitividade por sítios de infecção nodular em cultivares de soja. Agron Sulriograndense 16:205–219

    Google Scholar 

  25. 25.

    Ramos HJO, Souza EM, Soares-Ramos JRL, Pedrosa FO (2007) Determination of bean nodule occupancy by Rhizobium tropici using the double gfp and gusA genetic markers constitutively expressed from a new broad-host-range vector. World J Microbiol Biotechnol 23:713–717

    CAS  Article  Google Scholar 

  26. 26.

    Ruelle V, El Moualij B, Zorzi W, Leden TP, Pauw ED (2004) Rapid identification of environmental bacterial strains by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Rapid Commun Mass Spectrom 18:2013–2019

    CAS  Article  Google Scholar 

  27. 27.

    Sánchez-Juanes F, Ferreira L, Alonso de la Vega P, Valverde A, Barrios ML, Rivas R, Mateos PF, Martínez-Molina E, González-Buitrago JM, Trujillo ME, Velázquez E (2013) MALDI-TOF mass spectrometry as a tool for differentiation of Bradyrhizobium species: application to the identification of Lupinus nodulating strains. Syst Appl Microbiol 36:565–571

    Article  Google Scholar 

  28. 28.

    Siqueira AF, Ormeño-Orrillo E, Souza RC, Rodrigues EP, Almeida LGP, Barcellos FG (2014) Comparative genomics of Bradyrhizobium japonicum CPAC 15 and Bradyrhizobium diazoefficiens CPAC 7: elite model strains for understanding symbiotic performance with soybean. BMC Genomics 15:420

    Article  Google Scholar 

  29. 29.

    Somasegaran P, Hoben HJ (1994) Handbook for rhizobia: methods in legume-Rhizobium technology. Springer-Verlag, New York

    Book  Google Scholar 

  30. 30.

    Suarez S, Ferroni A, Lotz A, Jolley KA, Guerin P, Leto J, Dauphin B, Jamet A, Maiden MC, Nassif X, Armengaud J (2013) Ribosomal proteins as biomarkers for bacterial identification by mass spectrometry in the clinical microbiology laboratory. J Microbiol Methods 94:390–396

    CAS  Article  Google Scholar 

  31. 31.

    Torres D, Revale S, Obando M, Maroniche G, Paris G, Perticari A, Vazquez M, Wisniewski-Dyé F, Martínez-Abarca F, Cassán F (2015) Genome sequence of Bradyrhizobium japonicum E109, one of the most agronomically used nitrogen-fixing rhizobacteria in Argentina. Genome Announc 3:1566–1614

    Article  Google Scholar 

  32. 32.

    Van Berkum P, Elia P, Song Q, Eardly BD (2012) Development and application of a multilocus sequence analysis method for the identification of genotypes within genus Bradyrhizobium and for establishing nodule occupancy of soybean (Glycine max L. Merr). Mol Plant-Microbe Interact 25:321–220

    Article  Google Scholar 

  33. 33.

    Vargas MAT, Mendes IC, Suhet AR, Peres JRR (1993) Serological distribution of Bradyrhizobium japonicum from Brazilian “Cerrados” areas under soybean cultivation. Rev Microbiol 24:239–243

    Google Scholar 

  34. 34.

    Vargas MAT, Mendes IC, Hungria M (2001) Response of field grown bean [Phaseolus vulgaris (L)] to Rhizobium inoculation and N fertilization in two Cerrados soils. Biol Fertil Soils 32:228–233

    Article  Google Scholar 

  35. 35.

    Vincent JM (1970) A manual for the practical study at root-nodule bacteria. Blackwell Scientific Publ, Oxford

    Google Scholar 

  36. 36.

    Weber DF, Keyser HH, Uratsu SL (1989) Serological distribution of Bradyrhizobium japonicum from United States soybean production area. Agron J 81:786–789

    Article  Google Scholar 

  37. 37.

    Weiser A, Shceneider L, Jung J, Schubert S (2012) MALDI-TOF MS in microbiological diagnostics – identification of microorganisms and beyond (mini review). Appl Microbiol Biotechnol 93:965–974

    Article  Google Scholar 

  38. 38.

    Welker M, Moore ER (2011) Application of whole-cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in systematic microbiology. Syst Appl Microbiol 34:2–11

    CAS  Article  Google Scholar 

  39. 39.

    Williams TL, Andrzejewski D, Lay JO, Musser SM (2003) Experimental factors affecting the quality and reproducibility of MALDI TOF mass spectra obtained from whole bacteria cells. J Am Soc Mass Spectrom 14:342–351

    CAS  Article  Google Scholar 

  40. 40.

    Ziegler D, Pothier JF, Ardley J, Fossou RK, Pflüger V, Meyer S, Vogel G, Tonolla M, Howieson J, Reeve W, Perret X (2015) Ribosomal protein biomarkers provide root nodule bacterial identification by MALDI-TOF MS. Appl Microbiol Biotechnol 99:5547–5562

    CAS  Article  Google Scholar 

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Acknowledgments

The authors thank the Laboratory of Mass Spectrometry of the Embrapa Recursos Genéticos e Biotecnologia for the use of the mass spectrometer and Dr. Juaci Malaquias for his help in the statistics analysis. M. Hungria, I.C. Mendes, and L.P. Silva are research fellows from CNPq.

Funding

This work was supported by Embrapa (02.13.08.001.00.00) and INCT–Plant-Growth Promoting Microorganisms for Agricultural Sustainability and Environmental Responsibility (CNPq 465133/2014-4, Fundação Araucária-STI, CAPES).

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Correspondence to Luciano Paulino Silva.

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Rolim, L., Santiago, T.R., dos Reis Junior, F.B. et al. Identification of soybean Bradyrhizobium strains used in commercial inoculants in Brazil by MALDI-TOF mass spectrometry. Braz J Microbiol 50, 905–914 (2019). https://doi.org/10.1007/s42770-019-00104-3

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Keywords

  • Bradyrhizobium elkanii
  • Bradyrhizobium japonicum
  • Bradyrhizobium diazoefficiens
  • Biotyper
  • ClinPro Tools