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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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.
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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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
- Bradyrhizobium elkanii
- Bradyrhizobium japonicum
- Bradyrhizobium diazoefficiens
- ClinPro Tools