On the basis of the analysis of known methods of selection of highly active rhizobia strains, a strategy of primary screening of soybean nitrogen-fixing bacteria Bradyrhizobium japonicum based on the symbiotic properties under conditions of model pot experiments at natural light and temperature is presented. Soybeans of the Lisbon variety were inoculated with B. japonicum mutants obtained by transposon mutagenesis using plasmid vector pSUP5011::Tn5. The results of selection of rhizobia for the economic-valuable features (virulence, nodulation, nitrogen fixation activity, stimulation of growth of aboveground biomass) are presented. The differences between B. japonicum transposon mutants in the ability to nodules formation on the roots of the host plant, the dynamics and intensity of the assimilation of atmospheric nitrogen by soybean-rhizobia symbiotic systems are revealed. The expediency of selection of micro-symbionts was pointed out not only at the early period of the formation of symbiotic systems, but also at stages of the most active their functioning. This provides the possibility to select strains with different types of dynamics of nitrogen fixation activity as well as the most aggressive and high-virulent strains in the period of formation of symbiosis. Tn5 mutants with an improved symbiotic phenotype as compared to control strain Bradyrhizobium japonicum 634b were selected.
Keywords: soybean, symbiosis, nitrogen fixation, nodule bacteria, Bradyrhizobium japonicum, nitrogen, efficiency
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1. Kots, S.Ya., Morgun, V.V., Patyka, V.F., Malichenko, S.M., Mamenko, P.M., Kiriziy, D.A., Mykhalkiv, L.M., Beregovenko, S.K. & Melnykova, N.M. (2011). Biological nitrogen fixation: legume-rhizobial symbiosis (Vol. 2). Kyiv: Logos [in Russian].
2. Petrychenko, V.F. & Kots, S.Ya. (2014). Symbiotic system in modern agricultural manufacture. Bull. NAS Ukraine, 3, pp. 57-66. https://doi.org/10.15407/visn2014.03.057
3. Ryabukha, S.S., Chernyshenko, P.V. & Serikova, L.G. (2012).The effectiveness of the use of chemical mutagens in the selection of soybeans. Selektsiya i nasinnytstvo, 102, pp. 60-62 [in Ukrainian].
4. Aranjuelo, I., Arrese-Igor, C. & Molero, G. (2014). Nodule performance within a chan- ging environmental context. J. Plant Physiol., 171, No. 12, pp. 1076-1090. https://doi.org/10.1016/j.jplph.2014.04.002
5. Kots, S.Ya., Vorobey, N.A., Kyrychenko, O.V., Melnykova, N.N., Mykhalkiv, L.M. & Pukhtayevych, P.P. (2016). Microbiological preparations for agriculture. Institute of Plant Physiology and Genetics NAS of Ukraine. Kyiv: Logos, 48 p. [in Ukrainian].
6. Provorov, N.A. & Tikhonovich, I.A. (2014). Genetic and molecular basis of symbiotic adaptations. Biol. Bull. Reviews, 4, No. 6, pp. 211-226. https://doi.org/10.1134/S2079086414060061
7. Aghaei, K. & Komatsu, S. (2014). Crop and medicinal plants proteomics in response to salt stress. Fron. Plant Sci, 4, No. 8, pp. 1-9.
8. Provorov, N.A., Onishchuk, O.P., Yurgel, S.N., Kurchak, O.N., Chizhevskaya, E.P., Vorobyov, N.I., Zatovskaya, T.V. & Simarov, B.V. (2014). Construction of high effective symbiotic bacteria: Evolutionary models and genetic approaches. Genetika, 50, No. 11, pp. 1273-1285. doi: 10.7868/S0016675814110113. https://doi.org/10.7868/S0016675814110113
9. Spaink, G., Herman, P., Kondorosi, A. & Hooykaas, P. (2002). The Rhizobiaceae. Molecular Biology of Model Plant-Assosiated Bacteria. St. Peterburg [in Russian].
10. Rilfe, B.G., Greshoff, P.M. & Shine, J. (1980). Rapid screening for symbiotic mutants of Rhizobium and white clover. Plant Sci. Lett., 19, pp. 265-268. https://doi.org/10.1016/0304-4211(80)90082-6
11. Sirois, J.C. & Peterson, P.A. (1981). A rapid screening method for Rhizobium meliloti symbiotic nitrogenase activity. Can. J. Microbiol., 28, pp. 265-268. https://doi.org/10.1139/m82-037
12. Fedorov, S.N., Fokina, I.G. & Simarov, B.V. (1986). Evaluation of the symbiotic properties of alfalfa nodule bacteria in laboratory conditions. S.-Kh. Biology, No. 1, pp. 112-118 [in Russian].
13. Vorobey, N.A., Zaets, V.M. & Kots, S.Ya. (2012). Biotechnology of effective Tn5-mutants creation of clover nodule bacteria Rhizobium leguminosarum bv. trifolii. Biotechnologia, 5, No. 3, pp. 53-61 [in Ukrainian].
14. Grodzinskiy, A.M. & Grodzinskiy, D.M. (1964). Quick reference guide for plant physiology. Kyiv: Naukova dumka, 1964 [in Russian].
15. Hardy, R.W.F., Holsten, R.D., Jackson, E.K. & Burns, R.C. (1968). The acetylene-ethylene assay for N2-fixation: Laboratory and field evaluation. Plant Physiol., 43, No. 8, pp. 1185-1207. doi: https://doi.org/10.1104/pp.43.8.1185 https://doi.org/10.1104/pp.43.8.1185
16. Krutylo, D.V. & Zotov, V.S. (2015). Genotypic analysis of nodule bacteria nodulating soybean in soils of Ukraine. Russ. J. Genet.: Appl. Res., 5, No. 2, pp. 102-109. doi: https://doi.org/10.1134/S2079059715020057 https://doi.org/10.1134/S2079059715020057
17. Vorobey, N.A., Kots, S.Ya. & Mamenko, P.M. (2013). Realization of nitrogen fixation potential of Tn5-mutants Bradyrhizobium japonicum in symbiosis with soybean plants Biotechnologia Acta, 6, No. 5, p. 122-130 [in Ukrainian].
18. Provorov, N.A. & Vorobyov, N.I. (2014). Adaptive and progressive evolution of plant-microbial symbiosis. Russ. J. Genet.: Appl. Res., 4, No. 2, pp. 88-97. https://doi.org/10.1134/S2079059714020075
19. Bonaldi, K., Gourion, B., Fardoux, J., Hannibal, L., Cartieaux, F., Boursot, M., Vallenet, D., Chaintreuil, C., Prin, Y., Nouwen N. & Giraud, E. (2010). Large-scale transposon mutagenesis of photosynthetic Bradyrhizobium sp. strain ORS278 reveals new genetic loci putatively important for nod-independent symbiosis with Aeschynomene indica. Mol. Plant Microbe Interact., 23, pp. 760-770. doi: https://doi: 10.1094/ MPMI-23-6-0760